Oligonucleotides comprising a non-phosphate backbone linkage

ABSTRACT

One aspect of the present invention relates to a ribonucleoside substituted with a phosphonamidite group at the 3′-position. In certain embodiments, the phosphonamidite is an alkyl phosphonamidite. Another aspect of the present invention relates to a double-stranded oligonucleotide comprising at least one non-phosphate linkage. Representative non-phosphate linkages include phosphonate, hydroxylamine, hydroxylhydrazinyl, amide, and carbamate linkages. In certain embodiments, the non-phosphate linkage is a phosphonate linkage. In certain embodiments, a non-phosphate linkage occurs in only one strand. In certain embodiments, a non-phosphate linkage occurs in both strands. In certain embodiments, a ligand is bound to one of the oligonucleotide strands comprising the double-stranded oligonucleotide. In certain embodiments, a ligand is bound to both of the oligonucleotide strands comprising the double-stranded oligonucleotide. In certain embodiments, the oligonucleotide strands comprise at least one modified sugar moiety. Another aspect of the present invention relates to a single-stranded oligonucleotide comprising at least one non-phosphate linkage. Representative non-phosphate linkages include phosphonate, hydroxylamine, hydroxylhydrazinyl, amide, and carbamate linkages. In certain embodiments, the non-phosphate linkage is a phosphonate linkage. In certain embodiments, a ligand is bound to the oligonucleotide strand. In certain embodiments, the oligonucleotide comprises at least one modified sugar moiety.

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Application Ser. No. 60/584,061, filed Jun. 30, 2004; and U.S.Provisional Patent Application Ser. No. 60/614,528, filed Sep. 30, 2004;the contents of both of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Oligonucleotide compounds have important therapeutic applications inmedicine. Oligonucleotides can be used to silence genes that areresponsible for a particular disease. Gene-silencing prevents formationof a protein by inhibiting translation. Importantly, gene-silencingagents are a promising alternative to traditional small, organiccompounds that inhibit the function of the protein linked to thedisease. siRNA, antisense RNA, and micro-RNA are oligonucleotides thatprevent the formation of proteins by gene-silencing.

siRNA

RNA interference (RNAi) is an evolutionarily conserved gene silencingmechanism, originally discovered in studies of the nematodeCaenorhabditis elegans (Lee et al, Cell 75:843 (1993); Reinhart et al.,Nature 403:901 (2000)). It is triggered by introducing dsRNA into cellsexpressing the appropriate molecular machinery, which then degrades thecorresponding endogenous mRNA. The mechanism involves conversion ofdsRNA into short RNAs that direct ribonucleases to homologous mRNAtargets (summarized, Ruvkun, Science 2294:797 (2001)). This process isrelated to normal defense against viruses and the mobilization oftransposons.

Double-stranded ribonucleic acids (dsRNAs) are naturally rare and havebeen found only in certain microorganisms, such as yeasts or viruses.Recent reports indicate that dsRNAs are involved in phenomena ofregulation of expression, as well as in the initiation of the synthesisof interferon by cells (Declerq et al., Meth. Enzymol. 78:291 (1981);Wu-Li, Biol. Chem. 265:5470 (1990)). In addition, dsRNA has beenreported to have anti-proliferative properties, which makes it possiblealso to envisage therapeutic applications (Aubel et al., Proc. Natl.Acad. Sci., USA 88:906 (1991)). For example, synthetic dsRNA has beenshown to inhibit tumor growth in mice (Levy et al. Proc. Nat. Acad. Sci.USA, 62:357-361 (1969)), to be active in the treatment of leukemic mice(Zeleznick et al., Proc. Soc. Exp. Biol. Med. 130:126-128 (1969)); andto inhibit chemically-induced tumorigenesis in mouse skin (Gelboin etal., Science 167:205-207 (1970)).

Treatment with dsRNA has become an important method for analyzing genefunctions in invertebrate organisms. For example, Dzitoveva et al.showed, that RNAi can be induced in adult fruit flies by injecting dsRNAinto the abdomen of anesthetized Drosophila, and that this method canalso target genes expressed in the central nervous system (Mol.Psychiatry 6(6):665-670 (2001)). Both transgenes and endogenous geneswere successfully silenced in adult Drosophila by intra-abdominalinjection of their respective dsRNA. Moreover, Elbashir et al., providedevidence that the direction of dsRNA processing determines whether senseor antisense target RNA can be cleaved by a small interfering RNA(siRNA)-protein complex (Genes Dev. 15(2): 188-200 (2001)).

Two recent reports reveal that RNAi provides a rapid method to test thefunction of genes in the nematode Caenorhabditis elegans; and most ofthe genes on C. elegans chromosome I and III have now been tested forRNAi phenotypes (Barstead, Curr. Opin. Chem. Biol. 5(1):63-66 (2001);Tavernarakis, Nat. Genet. 24(2):180-183 (2000); Zamore, Nat. Struct.Biol. 8(9):746-750 (2001).). When used as a rapid approach to obtainloss-of-function information, RNAi was used to analyze a random set ofovarian transcripts and has identified 81 genes with essential roles inC. elegans embryogenesis (Piano et al., Curr. Biol. 10(24):1619-1622(2000). RNAi has also been used to disrupt the pupal hemocyte protein ofSarcophaga (Nishikawa et al., Eur. J. Biochem. 268(20):5295-5299(2001)).

Like RNAi in invertebrate animals, post-transcriptional gene silencing(PTGS) in plants is an RNA-degradation mechanism. In plants, this canoccur at both the transcriptional and the post-transcriptional levels;however, in invertebrates only post-transcriptional RNAi has beenreported to date (Bernstein et al., Nature 409(6818):295-296 (2001).Indeed, both involve double-stranded RNA (dsRNA), spread within theorganism from a localized initiating area, to correlate with theaccumulation of small interfering RNA (siRNA) and require putativeRNA-dependent RNA polymerases, RNA helicases and proteins of unknownfunctions containing PAZ and Piwi domains.

Some differences are evident between RNAi and PTGS were reported byVaucheret et al., J. Cell Sci. 114(Pt 17):3083-3091 (2001). First, PTGSin plants requires at least two genes—SGS3 (which encodes a protein ofunknown function containing a coil-coiled domain) and MET1 (whichencodes a DNA-methyltransferase)—that are absent in C. elegans, and thusare not required for RNAi. Second, all of the Arabidopsis mutants thatexhibit impaired PTGS are hyper-susceptible to infection by thecucumovirus CMV, indicating that PTGS participates in a mechanism forplant resistance to viruses. RNAi-mediated oncogene silencing has alsobeen reported to confer resistance to crown gall tumorigenesis (Escobaret al., Proc. Natl. Acad. Sci. USA, 98(23):13437-13442 (2001)).

RNAi is mediated by RNA-induced silencing complex (RISC), asequence-specific, multicomponent nuclease that destroys messenger RNAshomologous to the silencing trigger. RISC is known to contain short RNAs(approximately 22 nucleotides) derived from the double-stranded RNAtrigger, but the protein components of this activity remained unknown.Hammond et al. (Science 293(5532):1146-1150 (August 2001)) reportedbiochemical purification of the RNAi effector nuclease from culturedDrosophila cells, and protein microsequencing of a ribonucleoproteincomplex of the active fraction showed that one constituent of thiscomplex is a member of the Argonaute family of proteins, which areessential for gene silencing in Caenorhabditis elegans, Neurospora, andArabidopsis. This observation suggests links between the geneticanalysis of RNAi from diverse organisms and the biochemical model ofRNAi that is emerging from Drosophila in vitro systems.

Svoboda et al. reported in Development 127(19):4147-4156 (2000) thatRNAi provides a suitable and robust approach to study the function ofdormant maternal mRNAs in mouse oocytes. Mos (originally known as c-mos)and tissue plasminogen activator mRNAs are dormant maternal mRNAs arerecruited during oocyte maturation, and translation of Mos mRNA resultsin the activation of MAP kinase. The dsRNA directed towards Mos or TPAmRNAs in mouse oocytes specifically reduced the targeted mRNA in both atime- and concentration-dependent manner, and inhibited the appearanceof MAP kinase activity. See also, Svoboda et al. Biochem. Biophys. Res.Commun. 287(5):1099-1104 (2001).

Despite the advances in interference RNA technology, the need exists forsiRNA conjugates having improved pharmacologic properties. Inparticular, the oligonucleotide sequences have poor serum solubility,poor cellular distribution and uptake, and are rapidly excreted throughthe kidneys. It is known that oligonucleotides bearing the nativephospodiester (P═O) backbone are susceptable to nuclease-mediateddegradation. See L. L. Cummins et al. Nucleic Acids Res. 1995, 23, 2019.The stability of oligonucleotides has been increased by converting theP═O linkages to P═S linkages which are less susceptible to degradationby nucleases in vivo. Alternatively, the phosphate group can beconverted to a phosphoramidate which is less prone to enzymaticdegradation than the native phosphate. See Uhlmann, E.; Peyman, A. Chem.Rev. 1990, 90, 544. Modifications to the sugar groups of theoligonucleotide can confer stability to enzymatic degradation. Forexample, oligonucleotides comprising ribonucleic acids are less prone tonucleolytic degradation if the 2′-OH group of the sugar is converted toa methoxyethoxy group. See M. Manoharan Chem Bio Chem. 2002, 3, 1257 andreferences cited therein.

siRNA compounds are promising agents for a variety of diagnostic andtherapeutic purposes. siRNA compounds can be used to identify thefunction of a gene. In addition, siRNA compounds offer enormouspotential as a new type of pharmaceutical agent which acts by silencingdisease-causing genes. Research is currently underway to developinterference RNA therapeutic agents for the treatment of many diseasesincluding central-nervous-system diseases, inflammatory diseases,metabolic disorders, oncology, infectious diseases, and ocular disease.

Some progress has been made on increasing the cellular uptake ofsingle-stranded oligonucleotides, including increasing the membranepermeability via conjugates and cellular delivery of oligonucleotides.In U.S. Pat. No. 6,656,730, M. Manoharan describes compositions in whicha ligand that binds serum, vascular, or cellular proteins may beattached via an optional linking moiety to one or more sites on anoligonucleotide. These sites include one or more of, but are not limitedto, the 2′-position, 3′-position, 5′-position, the internucleotidelinkage, and a nucleobase atom of any nucleotide residue.

Antisense RNA

Antisense methodology is the complementary hybridization of relativelyshort oligonucleotides to mRNA or DNA such that the normal, essentialfunctions, such as protein synthesis, of these intracellular nucleicacids are disrupted. Hybridization is the sequence-specific hydrogenbonding via Watson-Crick base pairs of oligonucleotides to RNA orsingle-stranded DNA. Such base pairs are said to be complementary to oneanother.

The naturally-occurring events that provide the disruption of thenucleic acid function, discussed by Cohen (Oligonucleotides: AntisenseInhibitors of Gene Expression, CRC Press, Inc., 1989, Boca Raton, Fla.)are thought to be of two types. The first, hybridization arrest,describes the terminating event in which the oligonucleotide inhibitorbinds to the target nucleic acid and thus prevents, by simple sterichindrance, the binding of essential proteins, most often ribosomes, tothe nucleic acid. Methyl phosphonate oligonucleotides (Miller et al.(1987) Anti-Cancer Drug Design, 2:117-128), and α-anomeroligonucleotides are the two most extensively studied antisense agentswhich are thought to disrupt nucleic acid function by hybridizationarrest.

Another means by which antisense oligonucleotides disrupt nucleic acidfunction is by hybridization to a target mRNA, followed by enzymaticcleavage of the targeted RNA by intracellular RNase H. A2′-deoxyribofuranosyl oligonucleotide or oligonucleotide analoghybridizes with the targeted RNA and this duplex activates the RNase Henzyme to cleave the RNA strand, thus destroying the normal function ofthe RNA. Phosphorothioate oligonucleotides are the most prominentexample of an antisense agent that operates by this type of antisenseterminating event.

Considerable research is being directed to the application ofoligonucleotides and oligonucleotide analogs as antisense agents fordiagnostics, research applications and potential therapeutic purposes.One of the major hurdles that has only partially been overcome in vivois efficient cellular uptake which is severely hampered by the rapiddegradation and excretion of oligonucleotides. The generally acceptedprocess of cellular uptake is by receptor-mediated endocytosis which isdependent on the temperature and concentration of the oligonucleotidesin serum and extra vascular fluids.

Efforts aimed at improving the transmembrane delivery of nucleic acidsand oligonucleotides have utilized protein carriers, antibody carriers,liposomal delivery systems, electroporation, direct injection, cellfusion, viral vectors, and calcium phosphate-mediated transformation.However, many of these techniques are limited by the types of cells inwhich transmembrane transport is enabled and by the conditions neededfor achieving such transport. An alternative that is particularlyattractive for transmembrane delivery of oligonucleotides ismodification of the physicochemical properties of the oligonucleotide.

Micro-RNA

Micro-RNAs are a large group of small RNAs produced naturally inorganisms, at least some of which regulate the expression of targetgenes. Micro-RNAs are formed from an approximately 70 nucleotidesingle-stranded hairpin precursor transcript by Dicer. V. Ambros et al.Current Biology 2003, 13, 807. In many instances, the micro-RNA istranscribed from a portion of the DNA sequence that previously had noknown function. Micro-RNAs are not translated into proteins, but ratherbind to specific messenger RNAs blocking translation. It is thought thatmicro-RNAs base-pair imprecisely with their targets to inhibittranslation. Founding members of the micro-RNA family are let-7 andlin-4. The let-7 gene encodes a small, highly conserved RNA species thatregulates the expression of endogenous protein-coding genes during wormdevelopment. The active RNA species is transcribed initially as an ˜70nt precursor, which is post-transcriptionally processed into a mature˜21 nt form. Both let-7 and lin-4 are transcribed as hairpin RNAprecursors which are processed to their mature forms by Dicer enzyme.

The need exists for modified oligonucleotide compounds with improvedserum solubility, cellular distribution and uptake, and stability invivo. The oligonucleotide compounds of the invention comprisingnon-phosphate linkages fulfill this need and provide other relatedadvantages.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to a ribonucleosidesubstituted with a phosphonamidite group at the 3′-position. In certainembodiments, the phosphonamidite is an alkyl phosphonamidite. Anotheraspect of the present invention relates to a double-strandedoligonucleotide comprising at least one non-phosphate linkage.Representative non-phosphate linkages include phosphonate,hydroxylamine, hydroxylhydrazinyl, amide, and carbamate linkages. Incertain embodiments, the non-phosphate linkage is a phosphonate linkage.In certain embodiments, a non-phosphate linkage occurs in only onestrand. In certain embodiments, a non-phosphate linkage occurs in bothstrands. In certain embodiments, a ligand is bound to one of theoligonucleotide strands comprising the double-stranded oligonucleotide.In certain embodiments, a ligand is bound to both of the oligonucleotidestrands comprising the double-stranded oligonucleotide. In certainembodiments, the oligonucleotide strands comprise at least one modifiedsugar moiety. Another aspect of the present invention relates to asingle-stranded oligonucleotide comprising at least one non-phosphatelinkage. Representative non-phosphate linkages include phosphonate,hydroxylamine, hydroxylhydrazinyl, amide, and carbamate linkages. Incertain embodiments, the non-phosphate linkage is a phosphonate linkage.In certain embodiments, a ligand is bound to the oligonucleotide strand.In certain embodiments, the oligonucleotide comprises at least onemodified sugar moiety.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 depicts various oligonucleotides that are conjugated to a ligand.Note that NA is an oligonucleotide (or a nucleic acid) comprising ofeither RNA or DNA or chimeric RNA-DNA, DNA-RNA, RNA-DNA-RNA orDNA-RNA-DNA. In certain instances, at least one among R₁, R₂ and R₃ isaromatic or substituted aromatic, when R₁ is aromatic or substitutedaromatic, R₂ is either H or any organic substituent, and R₃ is either Hor any organic substituent.

FIG. 2 depicts various oligonucleotides that are conjugated to a ligand.Note: In certain instances, at least one among R₁, R₂ and R₃ is aromaticor substituted aromatic. For rows A-E: NA=DNA or RNA. For row A: racemicand R and S isomers. For rows B and C: racemic and all four stereoisomers (RR, RS, SR and SS). For rows D and E: R=H or OH.

FIG. 3 depicts various NA building blocks with a serinol linker (see rowA in FIG. 2) having aralkyl ligands linked through alkyl and PEGtethers. Each ligand shown is either racemic or optically enriched orpure R or S isomer.

FIG. 4 depicts various NA building blocks with a pyrrolidine linker (seerow B in FIG. 2) having aralkyl ligands linked through alkyl and PEGtethers. Each ligand shown is either racemic or optically enriched orpure R or S isomer.

FIG. 5 depicts various NA building blocks with a hydroxyprolinol linker(see row C in FIG. 2) having aralkyl ligands linked through alkyl andPEG tethers. Each ligand shown is either racemic or optically enrichedor pure R or S isomer.

FIG. 6 depicts various NA building blocks with a nucleoside linker (seerow D in FIG. 2) having aralkyl ligands linked through selected tethers.Each ligand shown is either racemic or optically enriched or pure R or Sisomer.

FIG. 7 depicts various NA building blocks with a nucleoside linker (seerow D in FIG. 2) having aralkyl ligands linked through selected tethers.Each ligand shown is either racemic or optically enriched or pure R or Sisomer.

FIG. 8 depicts various NA building blocks with a nucleoside linker (seerow E in FIG. 2) having aralkyl ligands linked through selected tethers.Each ligand shown is either racemic or optically enriched or pure R or Sisomer.

FIG. 9 depicts various NA building blocks with a nucleoside linker (seerow E in FIG. 2) having aralkyl ligands linked through selected tethers.Each ligand shown is either racemic or optically enriched or pure R or Sisomer.

FIG. 10 depicts various oligonucleotides that are conjugated to aligand. NA is an oligonucleotide (or a nucleic acid) comprising of RNAor DNA or chimeric RNA-DNA, DNA-RNA, RNA-DNA-RNA or DNA-RNA-DNA. Incertain instances, at least one among R₁, R₂ and R₃ is aromatic orsubstituted aromatic, when R₁ is aromatic or substituted aromatic, R₂ iseither H or any organic substituent and R₃ is either H or any organicsubstituent.

FIG. 11 depicts various siRNA duplexes conjugated with naproxen. I:Unmodified siRNA with overhang at the 3′-end of each strand. II: siRNAduplex with naproxen conjugation at the 3′-end of sense strand. III:siRNA duplex with naproxen conjugation at the 3′-end of antisensestrand. IV: siRNA duplex with naproxen conjugation at the 3′-end ofsense and antisense strands. V: siRNA with naproxen conjugation at the3′ and 5′-ends of sense strand. VI: siRNA duplex with naproxenconjugation at the 5′-end of sense strand. VII: siRNA duplex withnaproxen conjugation at the 5′-end of sense and 3′-end antisensestrands.

FIG. 12 depicts a procedure for solid-phase oligonucleotide synthesis.

FIG. 13 depicts results from denaturing gel analysis of the human serumstability assay for duplex 101 and 104 (See Example 14).

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention relates to a ribonucleosidesubstituted with a phosphonamidite group at the 3′-position. Thesecompounds can be used to prepare oligonucleotides used in gene therapy.Oligonucleosides prepared from 3′-phosphonamidite substitutednucleosides have phosphonate linkages which are less prone todegradation in vivo. In certain instances, the phosphonamidite is analkyl phosphonamidite. In addition, the 2′-position of theribonucleoside can be protected with a protecting group that can beeasily removed under mild conditions. One example of a protecting groupthat can be removed under mild conditions is a silyl protecting group.In a preferred embodiment, the protecting group istert-butyldimethylsilyl.

Another aspect of the present invention relates to a double-strandedoligonucleotide comprising at least one non-phosphate linkage. Thenon-phosphate moiety renders the oligonucleotide less prone todegradation in vivo. A large number of non-phosphate functional groupsare known in the art and are amenable to the present invention. Thenon-phosphate linkage can be a functional group that contains aphosphorous atom, or a functional group that does not contain aphosphorous atom. Representative non-phosphate linkages amenable to thepresent invention are phosphonate, hydroxylamine, hydroxylhydrazinyl,amide, and carbamate linkages. In certain embodiments, the non-phosphatelinkage is a phosphonate linkage. The non-phosphate linkage can occur inonly one strand or in both strands. In certain instances, there areabout 1-5 non-phosphate linkages per double-stranded oligonucleotide. Incertain instances, there are about 5-10 non-phosphate linkages perdouble-stranded oligonucleotide. In certain instances, there are about10-20 non-phosphate linkages per double-stranded oligonucleotide. Incertain instances, there are about 1-2 non-phosphate linkages per strandin the double-stranded oligonucleotide. In certain instances, there areabout 3-5 non-phosphate linkages per strand in the double-strandedoligonucleotide. In certain instances, there are about 5-10non-phosphate linkages per strand in the double-strandedoligonucleotide. In certain instances, there are about 10-15non-phosphate linkages per strand in the double-strandedoligonucleotide. A non-phosphate linkage can be located near theterminus of the oligonucleotide strand or in the interior of theoligonucleotide strand. In certain instances, a non-phosphate linkage islocated between the first and second nucleoside at the 3′-terminus ofthe oligonucleotide strand. In certain instances, a non-phosphatelinkage is located between the first and second nucleoside at the5′-terminus of the oligonucleotide strand. In certain instances, anon-phosphate linkage is located between the first and second nucleosideat the 3′-terminus of the oligonucleotide strand, and a non-phosphatelinkage is located between the first and second nucleoside at the5′-terminus of the oligonucleotide strand. In certain instances, thereare two adjacent non-phosphate linkages.

Additional examples of non-phosphate linkages include, for example,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiralphosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′, or 2′ to 2′ linkage.In certain instances, the oligonucleotides have inverted polaritycomprising a single 3′ to 3′ linkage at the 3′-most internucleotidelinkage i.e. a single inverted nucleoside residue which may be abasic(the nucleobase is missing or has a hydroxyl group in place thereof).Various salts, mixed salts and free acid forms are also included.

Representative examples of non-phosphate linkages that contain aphosphorus atom include phosphoramidate (—O—P(O)(NJ)-O—), phosphonate(—O—P(J)(O)—O—), thionophosphoramidate (—O—P(O)(NJ)-S—),thionoalkylphosphonate (—O—P(S)(J)-O—), thionoalkylphosphotriester(—O—P(O)(OJ)-S—), phosphoramidate (—N(J)-P(O)(O)—O—), andboranophosphate (—R—P(O)(O)-J-), wherein J denotes a substituent groupwhich is commonly hydrogen or an alkyl group or a more complicated group(e.g., aryl, aralkyl, cycloalkyl, heterocycloalkyl, alkenyl, and thelike) that varies from one type of linkage to another. RepresentativeUnited States patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697; and5,625,050; each of which is herein incorporated by reference.

Non-phosphate linkages that do not include a phosphorus atom includeshort-chain alkyl or cycloalkyl internucleoside linkages, mixedheteroatom and alkyl or cycloalkyl internucleoside linkages, or one ormore short-chain heteroatomic or heterocyclic internucleoside linkages.These include those having morpholino linkages (formed in part from thesugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxideand sulfone backbones; formacetyl and thioformacetyl backbones;methylene formacetyl and thioformacetyl backbones; riboacetyl backbones;alkene containing backbones; sulfamate backbones; methyleneimino andmethylenehydrazino backbones; sulfonate and sulfonamide backbones; amidebackbones; and others having mixed N, O, S and CH₂ components. Foradditional details, see Y. S. Sanghvi in Comprehensive Natural Products,Barton, B.; Nakanishi, K.; Meth-Coth, O.; and Kool, E. T. Eds.;Elsevier, N.Y., 1999, vol 7, 285 which is hereby incorporated byreference.

Representative non-phosphorus containing linkages include thiodiester(—O—C(O)—S—), thionocarbamate (—O—C(O)(NJ)-S—), siloxane (—O—Si(J)₂—O—),carbamate (—O—C(O)—NH— and —NH—C(O)—O—), sulfamate (—O—S(O)(O)—N— and—N—S(O)(O)—N—, morpholino sulfamide (—O—S(O)(N(morpholino)-),sulfonamide (—O—SO₂—NH—), sulfide (—CH₂—S—CH₂, sulfonate (—O—SO₂—CH₂—),N,N′-dimethylhydrazine (—CH₂—N(CH₃)—N(CH₃)—), thioformacetal(—S—CH₂—O—), formacetal (—O—CH₂—O—), thioketal (—S—C(J)₂—O—), ketal(—O—C(J)₂—O—), amine (—NH—CH₂—CH₂—), hydroxylamine (—CH₂—N(J)-O—),hydroxylimine (—CH═N—O—), and hydrazinyl (—CH₂—N(H)—N(H)—); wherein Jdenotes a substituent group which is commonly hydrogen or an alkyl groupor a more complicated group that varies from one type of linkage toanother. Representative United States patents that teach the preparationof the above oligonucleosides include, but are not limited to, U.S. Pat.Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269; and 5,677,439; each of whichis herein incorporated by reference.

Particularly preferred embodiments of the invention are oligonucleotideswith phosphorothioate backbones and phosponate backbones. In addition,oligonucleotides with phosphorothioate backbones and heteroatombackbones are preferred, and in particular —CH₂—NH—O—CH₂—,—CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone],—CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂——asdescribed in U.S. Pat. No. 5,489,677, and the amide backbones describedin U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides havingmorpholino backbone structures as described in U.S. Pat. No. 5,034,506.

In a preferred embodiment, the oligonucleotide is a ribonucleotidecomprising a non-phosphate linkage, and the non-phosphate linkage is aphosphorothioate, phosphorodithioate, boranophosphate,phosphorofluoridate, phosphoroselenoate, phosphoramidate,aminoalkylphosphonate, alkylphosphonate, phosphoramidate,phosphoramidimidate, phosphorotriester, phosphinate, amide, guanidine,urea, carbamate, thiocarbamate, amine, hydroxylamine, siloxane, sulfide,sulfone, sulfonate, sulfonamide, formacetal, thioformacetal, ether,alkyl, aryl, aralkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,heteroaryl, heteroaralkyl, alkenyl, alkynyl, acrylyl,dimethylhydrazinyl, hydroxyhydrazinyl, ketal, thioketal, or formacetal.

Another aspect of the present invention relates to a single-strandedoligonucleotide comprising at least one non-phosphate linkage.Representative non-phosphate linkages include phosphonate,hydroxylamine, hydroxylhydrazinyl, amide, and carbamate linkages. A morethorough listing of contemplated non-phosphate linkages is describedabove. In certain embodiments, the non-phosphate linkage is aphosphonate linkage. In certain embodiments, a ligand is bound to theoligonucleotide strand. In certain embodiments, the oligonucleotidecomprises at least one modified sugar moiety. In certain embodiments,the oligonucleotide is a ribonucleotide.

In a preferred embodiment, the single-stranded oligonucleotide is aribonucleotide comprising a non-phosphate linkage, and the non-phosphatelinkage is a phosphorothioate, phosphorodithioate, boranophosphate,phosphorofluoridate, phosphoroselenoate, phosphoramidate,aminoalkylphosphonate, alkylphosphonate, phosphoramidate,phosphoramidimidate, phosphorotriester, phosphinate, amide, guanidine,urea, carbamate, thiocarbamate, amine, hydroxylamine, siloxane, sulfide,sulfone, sulfonate, sulfonamide, formacetal, thioformacetal, ether,alkyl, aryl, aralkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,heteroaryl, heteroaralkyl, alkenyl, alkynyl, acrylyl,dimethylhydrazinyl, hydroxyhydrazinyl, ketal, thioketal, or formacetal.

Representative examples of oligonucleotides amenable to bothsingle-stranded and double-stranded oligonucleotides of the inventioncontaining one or more of alkylphosphonate, alkylthiophosphonate, andalkylphosphonate/alkylthiophosphonate backbone modifications are shownin the tables below. TABLE 1 Single incorporation of P-alkylphosphonatebackbone at the 3′-end of oligonucleotide

1. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = O2. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = S3. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n) = O,Y_(n+1) = S4. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂-Y_(n) = O,Y_(n+1) = O5. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n+1) = O6. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂-Y_(n+1) = S7. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁, Y₂ = S, Y₃-Y_(n) =O,Y_(n+1) = S8. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) S, Y₂,Y₄, Y₆ . . . Y_(n) = O9. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) S, Y₂,Y₄, Y₆ . . . Y_(n) = S10. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = O11. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = S12. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n) = O,Y_(n+1) = S13. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n) = O,Y_(n+1) = O14. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n+1) = O15. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n+1) = S16. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₂ = S, Y₃-Y_(n) =O, Y_(n+1) = S17. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) =S, Y₂, Y₄, Y₆ . . . Y_(n) = O18. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) =O, Y₂, Y₄, Y₆ . . . Y_(n) = S19. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = O20. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = S21. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n) = O,Y_(n+1) = S22. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂-Y_(n) = S,Y_(n+1) = O23. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n+1) = O24. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n+1) = S25. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₂ = S, Y₃-Y_(n) = O,Y_(n+1) = S26. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) = S,Y₂, Y₄, Y₆ . . . Y_(n) = O27. R₁, R₂ = OH, X = Me/isopropylltert-butyl, Y₁, Y₃ . . . Y_(n+1) = S,Y₂, Y₄, Y₆ . . . Y_(n) = S

TABLE 2 Single incorporation of P-alkylphosphonate backbone at the5′-end of oligonucleotide

1. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = O2. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = S3. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n) = O,Y_(n+1) = S4. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂-Y_(n) = O,Y_(n+1) = O5. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n+1) = O6. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂-Y_(n+1) = S7. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁, Y₂ = S, Y₃-Y_(n) =O,Y_(n+1) = S8. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) S, Y₂,Y₄, Y₆ . . . Y_(n) = O9. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) S, Y₂,Y₄, Y₆ . . . Y_(n) = S10. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = O11. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = S12. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n) = O,Y_(n+1) = S13. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n) = O,Y_(n+1) = O14. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n+1) = O15. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n+1) = S16. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₂ = S, Y₃-Y_(n) =O, Y_(n+1) = S17. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) =S, Y₂, Y₄, Y₆ . . . Y_(n) = O18. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) =O, Y₂, Y₄, Y₆ . . . Y_(n) = S19. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = O20. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = S21. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n) = O,Y_(n+1) = S22. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂-Y_(n) = S,Y_(n+1) = O23. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n+1) = O24. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n+1) = S25. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₂ = S, Y₃-Y_(n) = O,Y_(n+1) = S26. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) = S,Y₂, Y₄, Y₆ . . . Y_(n) = O27. R₁, R₂ = OH, X = Me/isopropylltert-butyl, Y₁, Y₃ . . . Y_(n+1) = S,Y₂, Y₄, Y₆ . . . Y_(n) = S

TABLE 3 Double incorporation of P-alkylphosphonate backbone at the3′-end of oligonucleotide

1. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = O2. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = S3. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n) = O,Y_(n+1) = S4. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂-Y_(n) = O,Y_(n+1) = O5. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n+1) = O6. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂-Y_(n+1) = S7. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁, Y₂ = S, Y₃-Y_(n) =O,Y_(n+1) = S8. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) S, Y₂,Y₄, Y₆ . . . Y_(n) = O9. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) S, Y₂,Y₄, Y₆ . . . Y_(n) = S10. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = O11. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = S12. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n) = O,Y_(n+1) = S13. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n) = O,Y_(n+1) = O14. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n+1) = O15. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n+1) = S16. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₂ = S, Y₃-Y_(n) =O, Y_(n+1) = S17. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) =S, Y₂, Y₄, Y₆ . . . Y_(n) = O18. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) =O, Y₂, Y₄, Y₆ . . . Y_(n) = S19. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = O20. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = S21. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n) = O,Y_(n+1) = S22. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂-Y_(n) = S,Y_(n+1) = O23. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n+1) = O24. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n+1) = S25. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₂ = S, Y₃-Y_(n) = O,Y_(n+1) = S26. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) = S,Y₂, Y₄, Y₆ . . . Y_(n) = O27. R₁, R₂ = OH, X = Me/isopropylltert-butyl, Y₁, Y₃ . . . Y_(n+1) = S,Y₂, Y₄, Y₆ . . . Y_(n) = S28. Xs can also be combinations of methyl and isopropyl or combinationsof methyl and tert-butyl or combinations of isopropyl and tert-butyl

TABLE 4 Double incorporation of P-alkylphosphonate backbone at the 3′-and 5′-end of oligonucleotide

1. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = O2. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = S3. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n) = O,Y_(n+1) = S4. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂-Y_(n) = O,Y_(n+1) = O5. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n+1) = O6. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂-Y_(n+1) = S7. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁, Y₂ = S, Y₃-Y_(n) =O,Y_(n+1) = S8. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) S, Y₂,Y₄, Y₆ . . . Y_(n) = O9. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) S, Y₂,Y₄, Y₆ . . . Y_(n) = S10. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = O11. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = S12. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n) = O,Y_(n+1) = S13. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n) = O,Y_(n+1) = O14. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n+1) = O15. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n+1) = S16. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₂ = S, Y₃-Y_(n) =O, Y_(n+1) = S17. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) =S, Y₂, Y₄, Y₆ . . . Y_(n) = O18. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) =O, Y₂, Y₄, Y₆ . . . Y_(n) = S19. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = O20. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = S21. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n) = O,Y_(n+1) = S22. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂-Y_(n) = S,Y_(n+1) = O23. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n+1) = O24. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n+1) = S25. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₂ = S, Y₃-Y_(n) = O,Y_(n+1) = S26. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) = S,Y₂, Y₄, Y₆ . . . Y_(n) = O27. R₁, R₂ = OH, X = Me/isopropylltert-butyl, Y₁, Y₃ . . . Y_(n+1) = S,Y₂, Y₄, Y₆ . . . Y_(n) = S28. Xs can also be combinations of methyl and isopropyl or combinationsof methyl and tert-butyl or combinations of isopropyl and tert-butyl

TABLE 5 Multiple Incorporation P-alkylphosphonate Backbone intoOligonucleotides.

1. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁-Y_(p+q+2) = O2. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁-Y_(p+q+2) = S3. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(p+q+1) = O,Y_(p+q+2) = S4. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂-Y_(p+q+1) = O,Y_(p+q+2) = O5. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(p+q+2) = O6. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂-Y_(p+q+2) = S7. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁, Y_(p+1), Y_(p+q+2) = S,Y₂-Y_(p), Y_(q)-Y_(p+q+1) = O8. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁, Y_(p+1), Y_(p+q+2) = O,Y₂-Y_(p), Y_(q)-Y_(p+q+1) = S9. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+1) = S, Y₁-Y_(p),Y_(q)-Y_(p+q+2) = O10. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+1) = S, Y₁-Y_(p),Y_(q)-Y_(p+q+2) = S11. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+q+2) = S,Y₁-Y_(p+q+1) = O12. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+q+2) = S,Y₁-Y_(p+q+1) = S13. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(p+q+2) = O14. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(p+q+2) = S15. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(p+q+1) =O, Y_(p+q+2) = S16. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(p+q+1) =O, Y_(p+q+2) = O17. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(p+q+2) =O18. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(p+q+2) =S19. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y_(p+1), Y_(p+q+2)= S, Y₂-Y_(p), Y_(q)-Y_(p+q+1) = O20. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y_(p+1), Y_(p+q+2)= S, Y₂-Y_(p), Y_(q)-Y_(p+q+1) = S21. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+1) = S, Y₁-Y_(p),Y_(q)-Y_(p+q+2) = O22. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+1) = S, Y₁-Y_(p),Y_(q)-Y_(p+q+2) = S23. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+q+2) = S,Y₁-Y_(p+q+1) = O24. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+q+2) = S,Y₁-Y_(p+q+1) = S25. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(p+q+2) = O26. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(p+q+2) = S27. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(p+q+1) = O,Y_(p+q+2) = S28. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂-Y_(p+q+1) = S,Y_(p+q+2) = O29. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(p+q+2) = O30. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂-Y_(p+q+2) = S31. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y_(p+1), Y_(p+q+2) =S, Y₂-Y_(p), Y_(q)-Y_(p+q+1) = O32. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y_(p+1), Y_(p+q+2) =S, Y₂-Y_(p), Y_(q)-Y_(p+q+1) = S33. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+1) = S, Y₁-Y_(p),Y_(q)-Y_(p+q+2) = O34. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+1) = S, Y₁-Y_(p),Y_(q)-Y_(p+q+2) = S35. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+q+2) = S,Y₁-Y_(p+q+1) = O36. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+q+2) = S,Y₁-Y_(p+q+1) = S37. Xs can also be combinations of methyl and isopropyl or combinationsof methyl and tert-butyl or combinations of methyl, isopropyl andtert-butyl or combinations of isopropyl and tert-butyl

TABLE 6 Multiple Incorporation P-alkylphosphonate Backbone intoOligonucleotides.

1. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁-Y_(p+q+2) = O2. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁-Y_(p+q+2) = S3. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(p+q+1) = O,Y_(p+q+2) = S4. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂-Y_(p+q+1) = O,Y_(p+q+2) = O5. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(p+q+2) = O6. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂-Y_(p+q+2) = S7. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁, Y_(p+1), Y_(p+q+2) = S,Y₂-Y_(p), Y_(q)-Y_(p+q+1) = O8. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁, Y_(p+1), Y_(p+q+2) = O,Y₂-Y_(p), Y_(q)-Y_(p+q+1) = S9. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+1) = S, Y₁-Y_(p),Y_(q)-Y_(p+q+2) = O10. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+1) = S, Y₁-Y_(p),Y_(q)-Y_(p+q+2) = S11. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+q+2) = S,Y₁-Y_(p+q+1) = O12. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+q+2) = S,Y₁-Y_(p+q+1) = S13. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(p+q+2) = O14. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(p+q+2) = S15. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(p+q+1) =O, Y_(p+q+2) = S16. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(p+q+1) =O, Y_(p+q+2) = O17. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(p+q+2) =O18. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(p+q+2) =S19. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y_(p+1), Y_(p+q+2)= S, Y₂-Y_(p), Y_(q)-Y_(p+q+1) = O20. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y_(p+1), Y_(p+q+2)= S, Y₂-Y_(p), Y_(q)-Y_(p+q+1) = S21. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+1) = S, Y₁-Y_(p),Y_(q)-Y_(p+q+2) = O22. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+1) = S, Y₁-Y_(p),Y_(q)-Y_(p+q+2) = S23. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+q+2) = S,Y₁-Y_(p+q+1) = O24. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+q+2) = S,Y₁-Y_(p+q+1) = S25. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(p+q+2) = O26. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(p+q+2) = S27. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(p+q+1) = O,Y_(p+q+2) = S28. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂-Y_(p+q+1) = S,Y_(p+q+2) = O29. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(p+q+2) = O30. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂-Y_(p+q+2) = S31. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y_(p+1), Y_(p+q+2) =S, Y₂-Y_(p), Y_(q)-Y_(p+q+1) = O32. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y_(p+1), Y_(p+q+2) =S, Y₂-Y_(p), Y_(q)-Y_(p+q+1) = S33. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+1) = S, Y₁-Y_(p),Y_(q)-Y_(p+q+2) = O34. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+1) = S, Y₁-Y_(p),Y_(q)-Y_(p+q+2) = S35. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+q+2) = S,Y₁-Y_(p+q+1) = O36. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+q+2) = S,Y₁-Y_(p+q+1) = S37. Xs can also be combinations of methyl and isopropyl or combinationsof methyl and tert-butyl or combinations of isopropyl and tert-butyl

TABLE 7 Multiple Incorporation P-alkylphosphonate Backbone intoOligonucleotides.

1. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁-Y_(p+q+2) = O2. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁-Y_(p+q+2) = S3. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(p+q+1) = O,Y_(p+q+2) = S4. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂-Y_(p+q+1) = O,Y_(p+q+2) = O5. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(p+q+2) = O6. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂-Y_(p+q+2) = S7. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁, Y_(p+1), Y_(p+q+2) = S,Y₂-Y_(p), Y_(q)-Y_(p+q+1) = O8. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁, Y_(p+1), Y_(p+q+2) = O,Y₂-Y_(p), Y_(q)-Y_(p+q+1) = S9. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+1) = S, Y₁-Y_(p),Y_(q)-Y_(p+q+2) = O10. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+1) = S, Y₁-Y_(p),Y_(q)-Y_(p+q+2) = S11. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+q+2) = S,Y₁-Y_(p+q+1) = O12. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+q+2) = S,Y₁-Y_(p+q+1) = S13. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(p+q+2) = O14. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(p+q+2) = S15. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(p+q+1) =O, Y_(p+q+2) = S16. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(p+q+1) =O, Y_(p+q+2) = O17. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(p+q+2) =O18. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(p+q+2) =S19. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y_(p+1), Y_(p+q+2)= S, Y₂-Y_(p), Y_(q)-Y_(p+q+1) = O20. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y_(p+1), Y_(p+q+2)= S, Y₂-Y_(p), Y_(q)-Y_(p+q+1) = S21. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+1) = S, Y₁-Y_(p),Y_(q)-Y_(p+q+2) = O22. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+1) = S, Y₁-Y_(p),Y_(q)-Y_(p+q+2) = S23. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+q+2) = S,Y₁-Y_(p+q+1) = O24. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+q+2) = S,Y₁-Y_(p+q+1) = S25. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(p+q+2) = O26. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(p+q+2) = S27. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(p+q+1) = O,Y_(p+q+2) = S28. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂-Y_(p+q+1) = S,Y_(p+q+2) = O29. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(p+q+2) = O30. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂-Y_(p+q+2) = S31. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y_(p+1), Y_(p+q+2) =S, Y₂-Y_(p), Y_(q)-Y_(p+q+1) = O32. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y_(p+1), Y_(p+q+2) =S, Y₂-Y_(p), Y_(q)-Y_(p+q+1) = S33. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+1) = S, Y₁-Y_(p),Y_(q)-Y_(p+q+2) = O34. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+1) = S, Y₁-Y_(p),Y_(q)-Y_(p+q+2) = S35. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+q+2) = S,Y₁-Y_(p+q+1) = O36. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+q+2) = S,Y₁-Y_(p+q+1) = S37. Xs can also be combinations of methyl and isopropyl or combinationsof methyl and tert-butyl or combinations of isopropyl and tert-butyl

TABLE 8 Oligonucleotide with P-alkylphosphonate backbone

1. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = O2. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = S3. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n) = O,Y_(n+1) = S4. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂-Y_(n) = O,Y_(n+1) = O5. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n+1) = O6. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂-Y_(n+1) = S7. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁, Y₂ = S, Y₃-Y_(n) =O,Y_(n+1) = S8. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) S, Y₂,Y₄, Y₆ . . . Y_(n) = O9. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) S, Y₂,Y₄, Y₆ . . . Y_(n) = S10. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = O11. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = S12. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n) = O,Y_(n+1) = S13. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n) = O,Y_(n+1) = O14. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n+1) = O15. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n+1) = S16. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₂ = S, Y₃-Y_(n) =O, Y_(n+1) = S17. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) =S, Y₂, Y₄, Y₆ . . . Y_(n) = O18. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) =O, Y₂, Y₄, Y₆ . . . Y_(n) = S19. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = O20. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = S21. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n) = O,Y_(n+1) = S22. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂-Y_(n) = S,Y_(n+1) = O23. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n+1) = O24. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n+1) = S25. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₂ = S, Y₃-Y_(n) = O,Y_(n+1) = S26. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) = S,Y₂, Y₄, Y₆ . . . Y_(n) = O27. R₁, R₂ = OH, X = Me/isopropylltert-butyl, Y₁, Y₃ . . . Y_(n+1) = S,Y₂, Y₄, Y₆ . . . Y_(n) = S28. Xs can also be combinations of methyl and isopropyl or combinationsof methyl and tert-butyl or combinations of methyl, isopropyl andtert-butyl or combinations of isopropyl and tert-butyl

TABLE 9 Single incorporation of P-alkylphosphonate backbone withα-anomer at the 3′-end of oligonucleotide

1. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = O2. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = S3. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n) = O,Y_(n+1) = S4. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂-Y_(n) = O,Y_(n+1) = O5. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n+1) = O6. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂-Y_(n+1) = S7. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁, Y₂ = S, Y₃-Y_(n) =O,Y_(n+1) = S8. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) S, Y₂,Y₄, Y₆ . . . Y_(n) = O9. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) S, Y₂,Y₄, Y₆ . . . Y_(n) = S10. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = O11. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = S12. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n) = O,Y_(n+1) = S13. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n) = O,Y_(n+1) = O14. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n+1) = O15. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n+1) = S16. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₂ = S, Y₃-Y_(n) =O, Y_(n+1) = S17. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) =S, Y₂, Y₄, Y₆ . . . Y_(n) = O18. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) =O, Y₂, Y₄, Y₆ . . . Y_(n) = S19. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = O20. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = S21. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n) = O,Y_(n+1) = S22. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂-Y_(n) = S,Y_(n+1) = O23. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n+1) = O24. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n+1) = S25. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₂ = S, Y₃-Y_(n) = O,Y_(n+1) = S26. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) = S,Y₂, Y₄, Y₆ . . . Y_(n) = O27. R₁, R₂ = OH, X = Me/isopropylltert-butyl, Y₁, Y₃ . . . Y_(n+1) = S,Y₂, Y₄, Y₆ . . . Y_(n) = S

TABLE 10 Single incorporation of P-alkylphosphonate backbone withα-anomer at the 5′-end of oligonucleotide

1. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = O2. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = S3. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n) = O,Y_(n+1) = S4. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂-Y_(n) = O,Y_(n+1) = O5. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n+1) = O6. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂-Y_(n+1) = S7. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁, Y₂ = S, Y₃-Y_(n) =O,Y_(n+1) = S8. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) S, Y₂,Y₄, Y₆ . . . Y_(n) = O9. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) S, Y₂,Y₄, Y₆ . . . Y_(n) = S10. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = O11. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = S12. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n) = O,Y_(n+1) = S13. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n) = O,Y_(n+1) = O14. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n+1) = O15. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n+1) = S16. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₂ = S, Y₃-Y_(n) =O, Y_(n+1) = S17. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) =S, Y₂, Y₄, Y₆ . . . Y_(n) = O18. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) =O, Y₂, Y₄, Y₆ . . . Y_(n) = S19. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = O20. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁-Y_(n+1) = S21. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n) = O,Y_(n+1) = S22. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂-Y_(n) = S,Y_(n+1) = O23. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n+1) = O24. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂-Y_(n+1) = S25. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₂ = S, Y₃-Y_(n) = O,Y_(n+1) = S26. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) = S,Y₂, Y₄, Y₆ . . . Y_(n) = O27. R₁, R₂ = OH, X = Me/isopropylltert-butyl, Y₁, Y₃ . . . Y_(n+1) = S,Y₂, Y₄, Y₆ . . . Y_(n) = S

TABLE 11 Double incorporation of P-alkylphosphonate backbone at the3′-end of oligonucleotide

1. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ − Y_(n+1) = O 2. R₁, R₂ =H, X = Me/isopropyl/tert-butyl, Y₁ − Y_(n+1) = S 3. R₁, R₂ = H, X =Me/isopropyl/tert-butyl, Y₁ = S, Y₂ − Y_(n) = O, Y_(n+1) = S 4. R₁, R₂ =H, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂ − Y_(n) = S, Y_(n+1) = O 5.R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂ − Y_(n+1) = O 6. R₁,R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂ − Y_(n+1) = S 7. R₁, R₂= H, X = Me/isopropyl/tert-butyl, Y₁, Y₂ = S, Y₃ − Y_(n) = O, Y_(n+1) =S 8. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) = S,Y₂, Y₄, Y₆ . . . Y_(n) = O 9. R₁, R₂ = H, X = Me/isopropyl/tert-butyl,Y₁, Y₃ . . . Y_(n+1) = O, Y₂, Y₄, Y₆ . . . Y_(n) = S 10. R₁ = H, R₂ =OH, X = Me/isopropyl/tert-butyl, Y₁ − Y_(n+1) = O 11. R₁ = H, R₂ = OH, X= Me/isopropyl/tert-butyl, Y₁ − Y_(n+1) = S 12. R₁ = H, R₂ = OH, X =Me/isopropyl/tert-butyl, Y₁ = S, Y₂ − Y_(n) = O, Y_(n+1) = S 13. R₁ = H,R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂ − Y_(n) = S, Y_(n+1) =O 14. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂ − Y_(n+1)= O 15. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂ −Y_(n+1) = S 16. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₂ =S, Y₃ − Y_(n) = O, Y_(n+1) = S 17. R₁ = H, R₂ = OH, X =Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) = S, Y₂, Y₄, Y₆ . . .Y_(n) = O 18. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . .Y_(n+1) = O, Y₂, Y₄, Y₆ . . . Y_(n) = S 19. R₁, R₂ = OH, X =Me/isopropyl/tert-butyl, Y₁ − Y_(n+1) = O 20. R₁, R₂ = OH, X =Me/isopropyl/tert-butyl, Y₁ − Y_(n+1) = S 21. R₁, R₂ = OH, X =Me/isopropyl/tert-butyl, Y₁ = S, Y₂ − Y_(n) = O, Y_(n+1) = S 22. R₁, R₂= OH, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂ − Y_(n) = S, Y_(n+1) = O23. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂ − Y_(n+1) = O24. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂ − Y_(n+1) = S25. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₂ = S, Y₃ − Y_(n) =O, Y_(n+1) = S 26. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . .. Y_(n+1) = S, Y₂, Y₄, Y₆ . . . Y_(n) = O 27. R₁, R₂ = OH, X =Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) = O, Y₂, Y₄, Y₆ . . .Y_(n) = S 28. Xs can also be combinations of methyl and isopropyl orcombinations of methyl and tert-butyl or combinations of isopropyl andtert-butyl

TABLE 12 Double incorporation of P-alkylphosphonate backbone withα-anomer at 3′- and 5′-end of oligonucleotide

1. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ − Y_(n+1) = O 2. R₁, R₂ =H, X = Me/isopropyl/tert-butyl, Y₁ − Y_(n+1) = S 3. R₁, R₂ = H, X =Me/isopropyl/tert-butyl, Y₁ = S, Y₂ − Y_(n) = O, Y_(n+1) = S 4. R₁, R₂ =H, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂ − Y_(n) = S, Y_(n+1) = O 5.R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ =S, Y₂ − Y_(n+1) = O 6. R₁,R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ =O, Y₂ − Y_(n+1) = S 7. R₁, R₂ =H, X = Me/isopropyl/tert-butyl, Y₁, Y₂ = S, Y₃ − Y_(n) = O, Y_(n+1) = S8. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) = S,Y₂, Y₄, Y₆ . . . Y_(n) = O 9. R₁, R₂ = H, X = Me/isopropyl/tert-butyl,Y₁, Y₃ . . . Y_(n+1) = O, Y₂, Y₄, Y₆ . . . Y_(n) = S 10. R₁ = H, R₂ =OH, X = Me/isopropyl/tert-butyl, Y₁ − Y_(n+1) = O 11. R₁ = H, R₂ = OH, X= Me/isopropyl/tert-butyl, Y₁ − Y_(n+1) = S 12. R₁ = H, R₂ = OH, X =Me/isopropyl/tert-butyl, Y₁ = S, Y₂ − Y_(n) = O, Y_(n+1) = S 13. R₁ = H,R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂ − Y_(n) = S, Y_(n+1) =O 14. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂ − Y_(n+1)= O 15. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂ −Y_(n+1) = S 16. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₂ =S, Y₃ − Y_(n) = O, Y_(n+1) = S 17. R₁ = H, R₂ = OH, X =Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) = S, Y₂, Y₄, Y₆ . . .Y_(n) = O 18. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . .Y_(n+1) = O, Y₂, Y₄, Y₆ . . . Y_(n) = S 19. R₁, R₂ = OH, X =Me/isopropyl/tert-butyl, Y₁ − Y_(n+1) = O 20. R₁, R₂ = OH, X =Me/isopropyl/tert-butyl, Y₁ − Y_(n+1) = S 21. R₁, R₂ = OH, X =Me/isopropyl/tert-butyl, Y₁ = S, Y₂ − Y_(n) = O, Y_(n+1) = S 22. R₁, R₂= OH, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂ − Y_(n) = S, Y_(n+1) = O23. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂ − Y_(n+1) = O24. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂ − Y_(n+1) = S25. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₂ = S, Y₃ − Y_(n) =O, Y_(n+1) = S 26. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . .. Y_(n+1) = S, Y₂, Y₄, Y₆ . . . Y_(n) = O 27. R₁, R₂ = OH, X =Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) = O, Y₂, Y₄, Y₆ . . .Y_(n) = S 28. Xs can also be combinations of methyl and isopropyl orcombinations of methyl and tert-butyl or combinations of isopropyl andtert-butyl

TABLE 13 Multiple Incorporation of P-alkylphosphonate Backbone withα-Anomer into Oligonucleotides.

1. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ − Y_(p+q+2) = O 2. R₁, R₂= H, X = Me/isopropyl/tert-butyl, Y₁ − Y_(p+q+2) = S 3. R₁, R₂ = H, X =Me/isopropyl/tert-butyl, Y₁ = S, Y₂ − Y_(p+q+1) = O, Y_(p+q+2) = S 4.R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂ − Y_(p+q+1) = S,Y_(p+q+2) = O 5. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ =S, Y₂ −Y_(p+q+2) = O 6. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ =O, Y₂ −Y_(p+q+2) = S 7. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁, Y_(p+1),Y_(p+q+2) = S, Y₂ − Y_(p), Y_(q) − Y_(p+q+1) = O 8. R₁, R₂ = H, X =Me/isopropyl/tert-butyl, Y₁, Y_(p+1), Y_(p+q+2) = O, Y₂ − Y_(p), Y_(q) −Y_(p+q+1) = S 9. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y_(p+1) = S,Y₁ − Y_(p), Y_(q) − Y_(p+q+2) = O 10. R₁, R₂ = H, X =Me/isopropyl/tert-butyl, Y_(p+1) = O, Y₁ − Y_(p), Y_(q) − Y_(p+q+2) = S11. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y_(p+q+2) = S, Y₁ −Y_(p+q+1) = O 12. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y_(p+q+2) =P, Y₁ − Y_(p+q+1) = S 13. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl,Y₁ − Y_(p+q+2) = O 14. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁− Y_(p+q+2) = S 15. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ =S, Y₂ − Y_(p+q+1) = O, Y_(p+q+2) = S 16. R₁ = H, R₂ = OH, X =Me/isopropyl/tert-butyl, Y₁ = O, Y₂ − Y_(p+q+1) = S, Y_(p+q+2) = O 17.R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂ − Y_(p+q+2) = O18. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂ − Y_(p+q+2)= S 19. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y_(p+1),Y_(p+q+2) = S, Y₂ − Y_(p), Y_(q) − Y_(p+q+1) = O 20. R₁ = H, R₂ = OH, X= Me/isopropyl/tert-butyl, Y₁, Y_(p+1), Y_(p+q+2) = O, Y₂ − Y_(p), Y_(q)− Y_(p+q+1) = S 21. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl,Y_(p+1) = S, Y₁ − Y_(p), Y_(q) − Y_(p+q+2) = O 22. R₁ = H, R₂ = OH, X =Me/isopropyl/tert-butyl, Y_(p+1) = O, Y₁ − Y_(p), Y_(q) − Y_(p+q+2) = S23. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+q+2) = S, Y₁ −Y_(p+q+1) = O 24. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl,Y_(p+q+2) = P, Y₁ − Y_(p+q+1) = S 25. R₁, R₂ = OH, X =Me/isopropyl/tert-butyl, Y₁ − Y_(p+q+2) = O 26. R₁, R₂ = OH, X =Me/isopropyl/tert-butyl, Y₁ − Y_(p+q+2) = S 27. R₁, R₂ = OH, X =Me/isopropyl/tert-butyl, Y₁ = S, Y₂ − Y_(p+q+1) = O, Y_(p+q+2) = S 28.R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂ − Y_(p+q+1) = S,Y_(p+q+2) = O 29. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂ −Y_(p+q+2) = O 30. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂ −Y_(p+q+2) = S 31. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y_(p+1),Y_(p+q+2) = S, Y₂ − Y_(p), Y_(q) − Y_(p+q+1) = O 32. R₁, R₂ = OH, X =Me/isopropyl/tert-butyl, Y₁, Y_(p+1), Y_(p+q+2) = O, Y₂ − Y_(p), Y_(q) −Y_(p+q+1) = S 33. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+1) = S,Y₁ − Y_(p), Y_(q) − Y_(p+q+2) = O 34. R₁, R₂ = OH, X =Me/isopropyl/tert-butyl, Y_(p+1) = O, Y₁ − Y_(p), Y_(q) − Y_(p+q+2) = S35. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+q+2) = S, Y₁ −Y_(p+q+1) = O 36. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+q+2) =P, Y₁ − Y_(p+q+1) = S 37. Xs can also be combinations of methyl andisopropyl or combinations of methyl and tert- butyl or combinations ofmethyl, isopropyl and tert-butyl or combinations of isopropyl andtert-butyl

TABLE 14 Multiple Incorporation of P-alkylphosphonate Backbone withα-Anomer into Oligonucleotides.

1. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ − Y_(p+q+2) = O 2. R₁, R₂= H, X = Me/isopropyl/tert-butyl, Y₁ − Y_(p+q+2) = S 3. R₁, R₂ = H, X =Me/isopropyl/tert-butyl, Y₁ = S, Y₂ − Y_(p+q+1) = O, Y_(p+q+2) = S 4.R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂ − Y_(p+q+1) = S,Y_(p+q+2) = O 5. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ =S, Y₂ −Y_(p+q+2) = O 6. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ =O, Y₂ −Y_(p+q+2) = S 7. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁, Y_(p+1),Y_(p+q+2) = S, Y₂ − Y_(p), Y_(q) − Y_(p+q+1) = O 8. R₁, R₂ = H, X =Me/isopropyl/tert-butyl, Y₁, Y_(p+1), Y_(p+q+2) = O, Y₂ − Y_(p), Y_(q) −Y_(p+q+1) = S 9. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y_(p+1) = S,Y₁ − Y_(p), Y_(q) − Y_(p+q+2) = O 10. R₁, R₂ = H, X =Me/isopropyl/tert-butyl, Y_(p+1) = O, Y₁ − Y_(p), Y_(q) − Y_(p+q+2) = S11. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y_(p+q+2) = S, Y₁ −Y_(p+q+1) = O 12. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y_(p+q+2) =P, Y₁ − Y_(p+q+1) = S 13. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl,Y₁ − Y_(p+q+2) = O 14. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁− Y_(p+q+2) = S 15. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ =S, Y₂ − Y_(p+q+1) = O, Y_(p+q+2) = S 16. R₁ = H, R₂ = OH, X =Me/isopropyl/tert-butyl, Y₁ = O, Y₂ − Y_(p+q+1) = S, Y_(p+q+2) = O 17.R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂ − Y_(p+q+2) = O18. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂ − Y_(p+q+2)= S 19. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y_(p+1),Y_(p+q+2) = S, Y₂ − Y_(p), Y_(q) − Y_(p+q+1) = O 20. R₁ = H, R₂ = OH, X= Me/isopropyl/tert-butyl, Y₁, Y_(p+1), Y_(p+q+2) = O, Y₂ − Y_(p), Y_(q)− Y_(p+q+1) = S 21. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl,Y_(p+1) = S, Y₁ − Y_(p), Y_(q) − Y_(p+q+2) = O 22. R₁ = H, R₂ = OH, X =Me/isopropyl/tert-butyl, Y_(p+1) = O, Y₁ − Y_(p), Y_(q) − Y_(p+q+2) = S23. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+q+2) = S, Y₁ −Y_(p+q+1) = O 24. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl,Y_(p+q+2) = P, Y₁ − Y_(p+q+1) = S 25. R₁, R₂ = OH, X =Me/isopropyl/tert-butyl, Y₁ − Y_(p+q+2) = O 26. R₁, R₂ = OH, X =Me/isopropyl/tert-butyl, Y₁ − Y_(p+q+2) = S 27. R₁, R₂ = OH, X =Me/isopropyl/tert-butyl, Y₁ = S, Y₂ − Y_(p+q+1) = O, Y_(p+q+2) = S 28.R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂ − Y_(p+q+1) = S,Y_(p+q+2) = O 29. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂ −Y_(p+q+2) = O 30. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂ −Y_(p+q+2) = S 31. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y_(p+1),Y_(p+q+2) = S, Y₂ − Y_(p), Y_(q) − Y_(p+q+1) = O 32. R₁, R₂ = OH, X =Me/isopropyl/tert-butyl, Y₁, Y_(p+1), Y_(p+q+2) = O, Y₂ − Y_(p), Y_(q) −Y_(p+q+1) = S 33. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+1) = S,Y₁ − Y_(p), Y_(q) − Y_(p+q+2) = O 34. R₁, R₂ = OH, X =Me/isopropyl/tert-butyl, Y_(p+1) = O, Y₁ − Y_(p), Y_(q) − Y_(p+q+2) = S35. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+q+2) = S, Y₁ −Y_(p+q+1) = O 36. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+q+2) =P, Y₁ − Y_(p+q+1) = S 37. Xs can also be combinations of methyl andisopropyl or combinations of methyl and tert- butyl or combinations ofisopropyl and tert-butyl

TABLE 15 Multiple Incorporation of P-alkylphosphonate Backbone withα-Anomer into Oligonucleotides.

1. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ − Y_(p+q+2) = O 2. R₁, R₂= H, X = Me/isopropyl/tert-butyl, Y₁ − Y_(p+q+2) = S 3. R₁, R₂ = H, X =Me/isopropyl/tert-butyl, Y₁ = S, Y₂ − Y_(p+q+1) = O, Y_(p+q+2) = S 4.R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂ − Y_(p+q+1) = S,Y_(p+q+2) = O 5. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ =S, Y₂ −Y_(p+q+2) = O 6. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ =O, Y₂ −Y_(p+q+2) = S 7. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁, Y_(p+1),Y_(p+q+2) = S, Y₂ − Y_(p), Y_(q) − Y_(p+q+1) = O 8. R₁, R₂ = H, X =Me/isopropyl/tert-butyl, Y₁, Y_(p+1), Y_(p+q+2) = O, Y₂ − Y_(p), Y_(q) −Y_(p+q+1) = S 9. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y_(p+1) = S,Y₁ − Y_(p), Y_(q) − Y_(p+q+2) = O 10. R₁, R₂ = H, X =Me/isopropyl/tert-butyl, Y_(p+1) = O, Y₁ − Y_(p), Y_(q) − Y_(p+q+2) = S11. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y_(p+q+2) = S, Y₁ −Y_(p+q+1) = O 12. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y_(p+q+2) =P, Y₁ − Y_(p+q+1) = S 13. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl,Y₁ − Y_(p+q+2) = O 14. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁− Y_(p+q+2) = S 15. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ =S, Y₂ − Y_(p+q+1) = O, Y_(p+q+2) = S 16. R₁ = H, R₂ = OH, X =Me/isopropyl/tert-butyl, Y₁ = O, Y₂ − Y_(p+q+1) = S, Y_(p+q+2) = O 17.R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂ − Y_(p+q+2) = O18. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂ − Y_(p+q+2)= S 19. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y_(p+1),Y_(p+q+2) = S, Y₂ − Y_(p), Y_(q) − Y_(p+q+1) = O 20. R₁ = H, R₂ = OH, X= Me/isopropyl/tert-butyl, Y₁, Y_(p+1), Y_(p+q+2) = O, Y₂ − Y_(p), Y_(q)− Y_(p+q+1) = S 21. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl,Y_(p+1) = S, Y₁ − Y_(p), Y_(q) − Y_(p+q+2) = O 22. R₁ = H, R₂ = OH, X =Me/isopropyl/tert-butyl, Y_(p+1) = O, Y₁ − Y_(p), Y_(q) − Y_(p+q+2) = S23. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+q+2) = S, Y₁ −Y_(p+q+1) = O 24. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl,Y_(p+q+2) = P, Y₁ − Y_(p+q+1) = S 25. R₁, R₂ = OH, X =Me/isopropyl/tert-butyl, Y₁ − Y_(p+q+2) = O 26. R₁, R₂ = OH, X =Me/isopropyl/tert-butyl, Y₁ − Y_(p+q+2) = S 27. R₁, R₂ = OH, X =Me/isopropyl/tert-butyl, Y₁ = S, Y₂ − Y_(p+q+1) = O, Y_(p+q+2) = S 28.R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂ − Y_(p+q+1) = S,Y_(p+q+2) = O 29. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂ −Y_(p+q+2) = O 30. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂ −Y_(p+q+2) = S 31. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y_(p+1),Y_(p+q+2) = S, Y₂ − Y_(p), Y_(q) − Y_(p+q+1) = O 32. R₁, R₂ = OH, X =Me/isopropyl/tert-butyl, Y₁, Y_(p+1), Y_(p+q+2) = O, Y₂ − Y_(p), Y_(q) −Y_(p+q+1) = S 33. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+1) = S,Y₁ − Y_(p), Y_(q) − Y_(p+q+2) = O 34. R₁, R₂ = OH, X =Me/isopropyl/tert-butyl, Y_(p+1) = O, Y₁ − Y_(p), Y_(q) − Y_(p+q+2) = S35. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+q+2) = S, Y₁ −Y_(p+q+1) = O 36. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y_(p+q+2) =P, Y₁ − Y_(p+q+1) = S 37. Xs can also be combinations of methyl andisopropyl or combinations of methyl and tert- butyl or combinations ofisopropyl and tert-butyl

TABLE 16 Oligonucleotide with P-alkylphosphonate backbone

1. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ − Y_(n+1) = O 2. R₁, R₂ =H, X = Me/isopropyl/tert-butyl, Y₁ − Y_(n+1) = S 3. R₁, R₂ = H, X =Me/isopropyl/tert-butyl, Y₁ = S, Y₂ − Y_(n) = O, Y_(n+1) = S 4. R₁, R₂ =H, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂ − Y_(n) = S, Y_(n+1) = O 5.R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ =S, Y₂ − Y_(n+1) = O 6. R₁,R₂ = H, X = Me/isopropyl/tert-butyl, Y₁ =O, Y₂ − Y_(n+1) = S 7. R₁, R₂ =H, X = Me/isopropyl/tert-butyl, Y₁, Y₂ = S, Y₃ − Y_(n) = O, Y_(n+1) = S8. R₁, R₂ = H, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) = S,Y₂, Y₄, Y₆ . . . Y_(n) = O 9. R₁, R₂ = H, X = Me/isopropyl/tert-butyl,Y₁, Y₃ . . . Y_(n+1) = O, Y₂, Y₄, Y₆ . . . Y_(n) = S 10. R₁ = H, R₂ =OH, X = Me/isopropyl/tert-butyl, Y₁ − Y_(n+1) = O 11. R₁ = H, R₂ = OH, X= Me/isopropyl/tert-butyl, Y₁ − Y_(n+1) = S 12. R₁ = H, R₂ = OH, X =Me/isopropyl/tert-butyl, Y₁ = S, Y₂ − Y_(n) = O, Y_(n+1) = S 13. R₁ = H,R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂ − Y_(n) = S, Y_(n+1) =O 14. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂ − Y_(n+1)= O 15. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂ −Y_(n+1) = S 16. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₂ =S, Y₃ − Y_(n) = O, Y_(n+1) = S 17. R₁ = H, R₂ = OH, X =Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) = S, Y₂, Y₄, Y₆ . . .Y_(n) = O 18. R₁ = H, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . . .Y_(n+1) = O, Y₂, Y₄, Y₆ . . . Y_(n) = S 19. R₁, R₂ = OH, X =Me/isopropyl/tert-butyl, Y₁ − Y_(n+1) = O 20. R₁, R₂ = OH, X =Me/isopropyl/tert-butyl, Y₁ − Y_(n+1) = S 21. R₁, R₂ = OH, X =Me/isopropyl/tert-butyl, Y₁ = S, Y₂ − Y_(n) = O, Y_(n+1) = S 22. R₁, R₂= OH, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂ − Y_(n) = S, Y_(n+1) = O23. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = S, Y₂ − Y_(n+1) = O24. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁ = O, Y₂ − Y_(n+1) = S25. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₂ = S, Y₃ − Y_(n) =O, Y_(n+1) = S 26. R₁, R₂ = OH, X = Me/isopropyl/tert-butyl, Y₁, Y₃ . .. Y_(n+1) = S, Y₂, Y₄, Y₆ . . . Y_(n) = O 27. R₁, R₂ = OH, X =Me/isopropyl/tert-butyl, Y₁, Y₃ . . . Y_(n+1) = O, Y₂, Y₄, Y₆ . . .Y_(n) = S 28. Xs can also be combinations of methyl and isopropyl orcombinations of methyl and tert-butyl or combinations of methyl,isopropyl and tert-butyl or combinations of isopropyl and tert-butyl

In certain instances, a ligand is bound to the oligonucleotide. Theligand improves the pharmacokinetic properties of the oligonucleotide.For double-stranded oligonucleotides, a ligand is bound to one of theoligonucleotide strands comprising the double-stranded oligonucleotidein certain instances. For double-stranded oligonucleotides, a ligand isbound to both of the oligonucleotide strands comprising thedouble-stranded oligonucleotide in certain instances. The ligand is anaromatic group, aralkyl group, or the radical of a steroid, bile acid,lipid, folic acid, pyridoxal, B12, riboflavin, biotin, polycycliccompound, crown ether, intercalator, cleaver molecule, protein-bindingagent, carbohydrate, or an optionally substituted saturated 5-memberedring. In certain embodiments, the ligand is an aralkyl group. Inaddition, the oligonucleotide may comprise a modified sugar moiety incertain instances. The sugar can be modified by replacing the2′-hydroxyl group with a fluorine atom or an —O-allyl group. Thismodification renders the oligonucleotide less prone to nucleolyticdegration.

For example, the present invention provides aralkyl-ligand-conjugatedsiRNA compounds that will impart improved pharmacokinetic properties tothe siRNA agent. Such compounds are prepared by covalently attaching anaralkyl ligand to siRNA. The aralkyl ligand, e.g., naproxen, improvesthe pharmacologic properties of the siRNA because the ligand bindsreversibly to one or more serum, vascular or cellular proteins. Thisreversible binding is expected to decrease urinary excretion, increaseserum half-life, and greatly increase the distribution of oligomericcompounds thus conjugated. In addition, the backbone of theoligonucleotide is modified to improve the stability of the siRNAcompound.

Conjugating a ligand to a siRNA can enhance its cellular absorption. Incertain instances, a hydrophobic ligand is conjugated to the siRNA tofacilitate direct permeation of the cellular membrane. Alternatively,the ligand conjugated to the siRNA is a substrate for receptor-mediatedendocytosis. These approaches have been used to facilitate cellpermeation of antisense oligonucleotides. For example, cholesterol hasbeen conjugated to various antisense oligonucleotides resulting incompounds that are substantially more active compared to theirnon-conjugated analogs. See M. Manoharan Antisense & Nucleic Acid DrugDevelopment 2002, 12, 103. Other lipophilic compounds that have beenconjugated to oligonucleotides include 1-pyrene butyric acid,1,3-bis-O-(hexadecyl)glycerol, and menthol. One example of a ligand forreceptor-mediated endocytosis is folic acid. Folic acid enters the cellby folate-receptor-mediated endocytosis. siRNA compounds bearing folicacid would be efficiently transported into the cell via thefolate-receptor-mediated endocytosis. Li and coworkers report thatattachment of folic acid to the 3′-terminus of an oligonucleotideresulted in an 8-fold increase in cellular uptake of theoligonucleotide. Li, S.; Deshmukh, H. M.; Huang, L. Pharm. Res. 1998,15, 1540. Other ligands that have been conjugated to oligonucleotidesinclude polyethylene glycols, carbohydrate clusters, cross-linkingagents, porphyrin conjugates, and delivery peptides.

In certain instances, conjugation of a cationic ligand tooligonucleotides often results in improved resistance to nucleases.Representative examples of cationic ligands are propylammonium anddimethylpropylammonium. Interestingly, antisense oligonucleotides werereported to retain their high binding affinity to mRNA when the cationicligand was dispersed throughout the oligonucleotide. See M. ManoharanAntisense & Nucleic Acid Drug Development 2002, 12, 103 and referencestherein.

The therapeutic effect of an oligonucleotide is realized when itinteracts with a specific cellular nucleic acid and effectively negatesits function. A preferred target is DNA or mRNA encoding a protein thatis responsible for a disease state. The overall effect of suchinterference with mRNA function is modulation of the expression of aprotein, wherein “modulation” means either an increase (stimulation) ora decrease (inhibition) in the expression of the protein. In the contextof the present invention, inhibition is the preferred form of modulationof gene expression. Nevertheless, the ultimate goal is to regulate theamount of such a protein.

To reach a target nucleic acid after administration, an oligonucleotideshould be able to overcome inherent factors such as rapid degradation inserum, short half-life in serum and rapid filtration by the kidneys withsubsequent excretion in the urine. Oligonucleotides that overcome theseinherent factors have increased serum half-life, distribution, cellularuptake and hence improved efficacy.

These enhanced pharmacokinetic parameters have been shown for selecteddrug molecules that bind plasma proteins (Olson and Christ, AnnualReports in Medicinal Chemistry, 1996, 31:327). Two proteins that havebeen studied more than most are human serum albumin (HSA) and α-1-acidglycoprotein. HSA binds a variety of endogenous and exogenous ligandswith association constants typically in the range of 10⁴ to 10⁶ M⁻¹.Association constants for ligands with α-1-acid glycoprotein are similarto those for HSA.

In a preferred embodiment of the invention, the protein targeted by theoligonucleotide is a serum protein. It is preferred that the serumprotein targeted by a conjugated oligomeric compound is animmunoglobulin (an antibody). Preferred immunoglobulins areimmunoglobulin G and immunoglobulin M. Immunoglobulins are known toappear in blood serum and tissues of vertebrate animals.

In another embodiment of the invention, the serum protein targeted bythe oligonucleotide is a lipoprotein. Lipoproteins are blood proteinshaving molecular weights generally above 20,000 that carry lipids andare recognized by specific c ell-surface receptors. The association withlipoproteins in the serum will initially increase pharmacokineticparameters such as half-life and distribution. A secondary considerationis the ability of lipoproteins to enhance cellular uptake viareceptor-mediated endocytosis.

In yet another embodiment, the serum protein targeted by theoligonucleotide compound is α-2-macroglobulin. In yet a furtherembodiment the serum protein targeted by the oligonucleotide isα-1-glycoprotein.

At least for therapeutic purposes, oligonucleotides should have a degreeof stability in serum to allow distribution and cellular uptake. Theprolonged maintenance of therapeutic levels of antisense agents in serumwill have a significant effect on the distribution and cellular uptakeand unlike conjugate groups that target specific cellular receptors, theincreased serum stability will effect all cells.

In the context of this invention, the siRNA comprises double-strandedoligonucleotides, wherein the term “oligonucleotide” refers to anoligomer or polymer of ribonucleic acid or deoxyribonucleic acid. Thisterm includes oligonucleotides composed of naturally-occurringnucleobases, sugars and covalent intersugar (backbone) linkages as wellas modified oligonucleotides having non-naturally-occurring portionswhich function similarly. Such modified or substituted oligonucleotidesare often preferred over native forms because of desirable propertiessuch as, for example, enhanced cellular uptake, enhanced binding totarget and increased stability in the presence of nucleases. Theoligonucleotides of the present invention preferably comprise from about5 to about 50 nucleosides. It is more preferred that sucholigonucleotides comprise from about 8 to about 30 nucleosides, with 15to 25 nucleosides being particularly preferred.

An oligonucleotide is a polymer of repeating units generically known asnucleotides or nucleosides. An unmodified (naturally occurring)nucleotide has three components: (1) a nitrogenous base linked by one ofits nitrogen atoms to (2) a 5-carbon cyclic sugar and (3) a phosphate,esterified to carbon 5 of the sugar. When incorporated into anoligonucleotide chain, the phosphate of a first nucleotide is alsoesterified to carbon 3 of the sugar of a second, adjacent nucleotide.The “backbone” of an unmodified oligonucleotide consists of (2) and (3),that is, sugars linked together by phosphodiester linkages between theCS (5′) position of the sugar of a first nucleotide and the C3 (3′)position of a second, adjacent nucleotide. A “nucleoside” is thecombination of (1) a nucleobase and (2) a sugar in the absence of aphosphate moiety (Kornberg, DNA Replication, W.H. Freeman & Co., SanFrancisco, 1980, pages 4-7). The backbone of an oligonucleotidepositions a series of bases in a specific order; the writtenrepresentation of this series of bases, which is conventionally writtenin 5′ to 3′ order, is known as a nucleotide sequence.

Oligonucleotides may comprise nucleoside or nucleotide sequencessufficient in identity and number to effect specific hybridization witha particular nucleic acid. Such oligonucleotides which specificallyhybridize to a portion of the sense strand of a gene are commonlydescribed as “antisense.” In the context of the invention,“hybridization” means hydrogen bonding, which may be Watson-Crick,Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementarynucleosides or nucleotides. For example, adenine and thymine arecomplementary nucleobases which pair through the formation of hydrogenbonds. “Complementary,” as used herein, refers to the capacity forprecise pairing between two nucleotides. For example, if a nucleotide ata certain position of an oligonucleotide is capable of hydrogen bondingwith a nucleotide at the same position of a DNA or RNA molecule, thenthe oligonucleotide and the DNA or RNA are considered to becomplementary to each other at that position. The oligonucleotide andthe DNA or RNA are complementary to each other when a sufficient numberof corresponding positions in each molecule are occupied by nucleotideswhich can hydrogen bond with each other. Thus, “specificallyhybridizable” and “complementary” are terms which are used to indicate asufficient degree of complementarity or precise pairing such that stableand specific binding occurs between the oligonucleotide and the DNA orRNA target. It is understood in the art that an oligonucleotide need notbe 100% complementary to its target DNA sequence to be specificallyhybridizable. An oligonucleotide is specifically hybridizable whenbinding of the oligonucleotide to the target DNA or RNA moleculeinterferes with the normal function of the target DNA or RNA to cause adecrease or loss of function, and there is a sufficient degree ofcomplementarity to a void non-specific binding of the oligonucleotide tonon-target sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment, or in the case of in vitro assays,under conditions in which the assays are performed.

The ligand-conjugated oligonucleotides of the invention can be preparedby attaching the ligand to the oligonucleotide through a monomer, e.g.,a chemically modified monomer that is integrated into theoligonucleotide agent. In a preferred embodiment, the coupling is by atether or a linker (or both) as described below, and the complex has theformula represented by:Ligand-[linker]_(optional)-[tether]_(optional)-oligonucleotide agent

While, in most cases, embodiments are described with respect to anoligonucleotide agent including a number of nucleotides, the inventionalso includes monomeric subunits having the structure:Ligand-[linker]_(optional)-[tether]_(optional)-monomer

Methods of making and incorporating the monomers into theoligonucleotide agents and methods of using those agents are included inthe invention. In preferred embodiments, the sugar, e.g., the ribosesugar of one or more of the nucleotides, (e.g., ribonucleotide,deoxynucleotide, or modified nucleotide) subunits of an oligonucleotideagent can be replaced with another moiety, e.g., a non-carbohydratecarrier. In certain instances, the non-carbohydrate is cyclic. Anucleotide subunit in which the sugar of the subunit has been soreplaced is referred to herein as a sugar replacement modificationsubunit (SRMS). This is often referred to as a tether. A cyclic carriermay be a carbocyclic ring system, i.e., all ring atoms are carbon atomsor a heterocyclic ring system, i.e., one or more ring atoms may be aheteroatom, e.g., nitrogen, oxygen, or sulfur. The cyclic carrier may bea monocyclic ring system, or may contain two or more rings, e.g. fusedrings. The cyclic carrier may be a fully saturated ring system, or itmay contain one or more double bonds.

The oligonucleotide agents of the invention include nucleic acidtargeting (NAT) oligonucleotide agents and protein-targeting (PT)oligonucleotide agents. NAT and PT oligonucleotide agents refer tosingle-stranded oligomers or polymers of ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA) or combined (chimeric) modifications of DNAand RNA. This term includes oligonucleotides composed of naturallyoccurring nucleobases, sugars, and covalent internucleoside (backbone)linkages as well as oligonucleotides having non-naturally-occurringportions that function similarly. Such modified or substitutedoligonucleotides are often preferred over native forms because ofdesirable properties such as enhanced cellular uptake, enhanced affinityfor nucleic acid target, and/or increased stability in the presence ofnucleases. NATs designed to bind to specific RNA or DNA targets havesubstantial complementarity, e.g., at least 70, 80, 90, or 100%complementary, with at least 10, 20, or 30 or more bases of a targetnucleic acid, and include antisense RNAs, miRNAs, and other non-duplexstructures which can modulate expression. Other NAT oligonucleotideagents include external guide sequence (EGS) oligonucleotides(oligozymes), DNAzymes, and ribozymes. These NATs may or may not bindvia Watson-Crick complementarity to their targets. PT oligonucleotideagents bind to protein targets, preferably by virtue ofthree-dimensional interactions, and modulate protein activity. Theyinclude decoy RNAs, aptamers, and the like.

The single-stranded oligonucleotide compounds of the inventionpreferably comprise from about 8 to about 50 nucleobases (i.e. fromabout 8 to about 50 linked nucleosides). NAT oligonucleotide agents arepreferably about 15 nucleotides long, or more preferably about 30nucleotides long. PT oligonucleotide agents are preferably about 18nucleotides long, or more preferably about 23 nucleotides long.Particularly preferred compounds are miRNAs and antisenseoligonucleotides, even more preferably those comprising from about 12 toabout 30 nucleobases.

While not wishing to be bound by theory, an oligonucleotide agent mayact by one or more of a number of mechanisms, including acleavage-dependent or cleavage-independent mechanism. A cleavage-basedmechanism can be RNAse H dependent and/or can include RISC complexfunction. Cleavage-independent mechanisms include occupancy-basedtranslational arrest, such as is mediated by miRNAs, or binding of theoligonucleotide agent to a protein, as do aptamers. Oligonucleotideagents may also be used to alter the expression of genes by changing thechoice of the splice site in a pre-mRNA. Inhibition of splicing can alsoresult in degradation of the improperly processed message, thusdown-regulating gene expression. Kole and colleagues (Sierakowska, etal. Proc. Natl. Acad. Sci. USA, 1996, 93:12840-12844) showed that2′-O-Me phosphorothioate oligonucleotides could correct aberrantbeta-globin splicing in a cellular system. Fully modified2′-methoxyethyl oligonucleotides and peptide nucleic acids (PNAs) wereable to redirect splicing of IL-5 receptor-α pre-mRNA (Karras et al.,Mol. Pharmacol. 2000, 58:380-387; Karras, et al., Biochemistry 2001,40:7853-7859).

MicroRNAs

The oligonucleotide agents include microRNAs (miRNAs). MicroRNAs aresmall noncoding RNA molecules that are capable of causingpost-transcriptional silencing of specific genes in cells such as by theinhibition of translation or through degradation of the targeted mRNA. AmiRNA can be completely complementary or can have a region ofnoncomplementarity with a target nucleic acid, consequently resulting ina “bulge” at the region of non-complementarity. The region ofnon-complementarity (the bulge) can be flanked by regions of sufficientcomplementarity, preferably complete complementarity to allow duplexformation. Preferably, the regions of complementarity are at least 8 to10 nucleotides long (e.g., 8, 9, or 10 nucleotides long). A miRNA caninhibit gene expression by repressing translation, such as when themicroRNA is not completely complementary to the target nucleic acid, orby causing target RNA degradation, which is believed to occur only whenthe miRNA binds its target with perfect complementarity. The inventionalso includes double-stranded precursors of miRNAs that may or may notform a bulge when bound to their targets.

A miRNA or pre-miRNA can be about 18-100 nucleotides in length, and morepreferably from about 18-80 nucleotides in length. Mature miRNAs canhave a length of about 19-30 nucleotides, preferably about 21-25nucleotides, particularly 21, 22, 23, 24, or 25 nucleotides. MicroRNAprecursors can have a length of about 70-100 nucleotides and have ahairpin conformation. MicroRNAs can be generated in vivo from pre-miRNAsby enzymes called Dicer and Drosha that specifically process longpre-miRNA into functional miRNA. The microRNAs or precursor miRNAsfeatured in the invention can be synthesized in vivo by a cell-basedsystem or can be chemically synthesized. MicroRNAs can be synthesized toinclude a modification that imparts a desired characteristic. Forexample, the modification can improve stability, hybridizationthermodynamics with a target nucleic acid, targeting to a particulartissue or cell-type, or cell permeability, e.g., by anendocytosis-dependent or -independent mechanism. Modifications can alsoincrease sequence specificity, and consequently decrease off-sitetargeting. Methods of synthesis and chemical modifications are describedin greater detail below.

In particular, an miRNA or a pre-miRNA featured in the invention canhave a chemical modification on a nucleotide in an internal (i.e.,non-terminal) region having noncomplementarity with the target nucleicacid. For example, a modified nucleotide can be incorporated into theregion of a miRNA that forms a bulge. The modification can include aligand attached to the miRNA, e.g., by a linker. The modification can,for example, improve pharmacokinetics or stability of a therapeuticmiRNA, or improve hybridization properties (e.g., hybridizationthermodynamics) of the miRNA to a target nucleic acid. In someembodiments, it is preferred that the orientation of a modification orligand incorporated into or tethered to the bulge region of a miRNA isoriented to occupy the space in the bulge region. This orientationfacilitates the improved hybridization properties or an otherwisedesired characteristic of the miRNA. For example, the modification caninclude a modified base or sugar on the nucleic acid strand or a ligandthat functions as an intercalator. These are preferably located in thebulge. The intercalator can be an aromatic, e.g., a polycyclic aromaticor heterocyclic aromatic compound. A polycyclic intercalator can havestacking capabilities, and can include systems with 2, 3, or 4 fusedrings. Universal bases can also be incorporated into the miRNAs.

In one embodiment, an miRNA or a pre-miRNA can include an aminoglycosideligand, which can cause the miRNA to have improved hybridizationproperties or improved sequence specificity. Exemplary aminoglycosidesinclude glycosylated polylysine; galactosylated polylysine; neomycin B;tobramycin; kanamycin A; and acridine conjugates of aminoglycosides,such as Neo-N-acridine, Neo-S-acridine, Neo-C-acridine,Tobra-N-acridine, and KanaA-N-acridine. Use of an acridine analog canincrease sequence specificity. For example, neomycin B has a highaffinity for RNA as compared to DNA, but low sequence-specificity.Neo-S-acridine, an acridine analog, has an increased affinity for theHIV Rev-response element (RRE). In some embodiments, the guanidineanalog (the guanidinoglycoside) of an aminoglycoside ligand is tetheredto an oligonucleotide agent. In a guanidinoglycoside, the amine group onthe amino acid is exchanged for a guanidine group. Attachment of aguanidine analog can enhance cell permeability of an oligonucleotideagent.

In one embodiment, the ligand can include a cleaving group thatcontributes to target gene inhibition by cleavage of the target nucleicacid. Preferably, the cleaving group is tethered to the miRNA in amanner such that it is positioned in the bulge region, where it canaccess and cleave the target RNA. The cleaving group can be, forexample, a bleomycin (e.g., bleomycin-A₅, bleomycin-A₂, orbleomycin-B₂), pyrene, phenanthroline (e.g., O-phenanthroline), apolyamine, a tripeptide (e.g., lys-tyr-lys tripeptide), or metal ionchelating group. The metal ion chelating group can include, e.g., anLu(III) or EU(III) macrocyclic complex, a Zn(II)2,9-dimethylphenanthroline derivative, a Cu(I) terpyridine, or acridine,which can promote the selective cleavage of target RNA at the site ofthe bulge by free metal ions, such as Lu(M). In some embodiments, apeptide ligand can be tethered to a miRNA or a pre-miRNA to promotecleavage of the target RNA, such as at the bulge region. For example,1,8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane (cyclam) can beconjugated to a peptide (e.g., by an amino acid derivative) to promotetarget RNA cleavage. The methods and compositions featured in theinvention include miRNAs that inhibit target gene expression by acleavage or non-cleavage dependent mechanism.

A miRNA or a pre-miRNA can be designed and synthesized to include aregion of noncomplementarity (e.g., a region that is 3, 4, 5, or 6nucleotides long) flanked by regions of sufficient complementarity toform a duplex (e.g., regions that are 7, 8, 9, 10, or 11 nucleotideslong). For increased nuclease resistance and/or binding affinity to thetarget, the miRNA sequences can include 2′-O-methyl, 2′-fluorine,2′-O-methoxyethyl, 2′-O-aminopropyl, 2′-amino, and/or phosphorothioatelinkages. The inclusion of furanose sugars in the oligonucleotidebackbone can also decrease endonucleolytic cleavage. An miRNA or apre-miRNA can be further modified by including a 3′-cationic group, orby inverting the nucleoside at the 3′-terminus with a 3′-3′ linkage. Inanother alternative, the 3′-terminus can be blocked with an aminoalkylgroup, e.g., a 3′-C5-aminoalkyl dT. Other 3′-conjugates can inhibit3′-5′ exonucleolytic cleavage. While not being bound by theory, a3′-conjugate, such as naproxen or ibuprofen, may inhibit exonucleolyticcleavage by sterically blocking the exonuclease from binding to the3′-end of oligonucleotide. Even small alkyl chains, aryl groups, orheterocyclic conjugates or modified sugars (D-ribose, deoxyribose,glucose etc.) can block 3′-5′-exonucleases.

In one embodiment, a miRNA or a pre-miRNA includes a modification thatimproves targeting, e.g. a targeting modification described above.Examples of modifications that target miRNA molecules to particular celltypes include carbohydrate sugars such as galactose,N-acetylgalactosamine, mannose; vitamins such as folates; other ligandssuch as RGDs and RGD mimics; and small molecules including naproxen,ibuprofen or other known protein-binding molecules.

A miRNA or a pre-miRNA can be constructed using chemical synthesisand/or enzymatic ligation reactions using procedures known in the art.For example, a miRNA or a pre-miRNA can be chemically synthesized usingnaturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between the miRNAor a pre-miRNA and target nucleic acids, e.g., phosphorothioatederivatives and acridine substituted nucleotides can be used. Otherappropriate nucleic acid modifications are described herein.Alternatively, the miRNA or pre-miRNA nucleic acid can be producedbiologically using an expression vector into which a nucleic acid hasbeen subcloned in an antisense orientation, i.e., RNA transcribed fromthe inserted nucleic acid will be of an antisense orientation to atarget nucleic acid of interest.

Antisense Nucleic Acid Sequences

The single-stranded oligonucleotide agents featured in the inventioninclude antisense nucleic acids. An “antisense” nucleic acid includes anucleotide sequence that is complementary to a “sense” nucleic acidencoding a gene expression product, e.g., complementary to the codingstrand of a double-stranded cDNA molecule or complementary to an RNAsequence, e.g., a pre-mRNA, mRNA, miRNA, or pre-miRNA. Accordingly, anantisense nucleic acid can form hydrogen bonds with a sense nucleic acidtarget.

Given a coding strand sequence such as the sequence of a sense strand ofa cDNA molecule, antisense nucleic acids can be designed according tothe rules of Watson and Crick base pairing. The antisense nucleic acidmolecule can be complementary to a portion of the coding or noncodingregion of an RNA, e.g., a pre-mRNA or mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of a pre-mRNA or mRNA, e.g., the 5′ UTR. Anantisense oligonucleotide can be about 10 to 25 nucleotides in length(e.g., 11, 12, 13, 14, 15, 16, 18, 19, 20, 21, 22, 23, or 24 nucleotidesin length). An antisense oligonucleotide can also be complementary to amiRNA or pre-miRNA.

An antisense nucleic acid can be constructed using chemical synthesisand/or enzymatic ligation reactions using procedures known in the art.For example, an antisense nucleic acid can be chemically synthesizedusing naturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between theantisense and target nucleic acids, e.g., phosphorothioate derivativesand acridine substituted nucleotides can be used. Other appropriatenucleic acid modifications are described herein. Alternatively, theantisense nucleic acid can be produced biologically using an expressionvector into which a nucleic acid has been subcloned in an antisenseorientation, i.e., RNA transcribed from the inserted nucleic acid willbe of an antisense orientation to a target nucleic acid of interest.

An antisense agent can include ribonucleotides only,deoxyribonucleotides only (e.g., oligodeoxynucleotides), or bothdeoxyribonucleotides and ribonucleotides. For example, an antisenseagent consisting only of ribonucleotides can hybridize to acomplementary RNA, and prevent access of the translation machinery tothe target RNA transcript, thereby preventing protein synthesis. Anantisense molecule including only deoxyribonucleotides, ordeoxyribonucleotides and ribonucleotides, e.g., DNA sequence flanked byRNA sequence at the 5′ and 3′ ends of the antisense agent, can hybridizeto a complementary RNA, and the RNA target can be subsequently cleavedby an enzyme such as RNAse H. Degradation of the target RNA preventstranslation. The flanking RNA sequences can include 2′-O-methylatednucleotides, and phosphorothioate linkages, and the internal DNAsequence can include phosphorothioate internucleotide linkages. Theinternal DNA sequence is preferably at least five nucleotides in lengthwhen targeting by RNAse H activity is desired.

For increased nuclease resistance, an antisense agent can be furthermodified by inverting the nucleoside at the 3′-terminus with a 3′-3′linkage. In another alternative, the 3′-terminus can be blocked with anaminoalkyl group. In certain instances, the antisense oligonucleotideagent includes a modification that improves targeting, e.g. a targetingmodification.

Decoy Nucleic Acids

An oligonucleotide agent featured in the invention can be a decoynucleic acid such as decoy RNA. A decoy nucleic acid resembles a naturalnucleic acid, but is modified to inhibit or interrupt the activity ofthe natural nucleic acid. For example, a decoy RNA can mimic the naturalbinding domain for a ligand, and compete with natural binding target forthe binding of a specific ligand. It has been shown that over-expressionof HIV trans-activation response (TAR) RNA can act as a “decoy” andefficiently bind HIV tat protein, thereby preventing it from binding toTAR sequences encoded in the HIV RNA. In one embodiment, a decoy RNAincludes a modification that improves targeting. The chemicalmodifications described above for miRNAs and antisense RNAs, anddescribed elsewhere herein, are also appropriate for use in decoynucleic acids.

Aptamers

Oligonucleotide agents of the invention also include aptamers. Anaptamer binds to a non-nucleic acid ligand, such as a small organicmolecule or protein, e.g., a transcription or translation factor, andsubsequently modifies its activity. An aptamer can fold into a specificstructure that directs the recognition of the targeted binding site onthe non-nucleic acid ligand. An aptamer can contain any of themodifications described herein. In certain instances, the aptamerincludes a modification that improves targeting, e.g., a targetingmodification. The chemical modifications described above for miRNAs andantisense RNAs, and described elsewhere herein, are also appropriate foruse in decoy nucleic acids.

Additional Features of the Oligonucleotides of the Invention

An oligonucleotide agent that is NAT (“nucleic acid targeting”) includesa region of sufficient complementarity to the target gene, and is ofsufficient length in terms of nucleotides, such that the oligonucleotideagent forms a duplex with the target nucleic acid. The oligonucleotideagent can modulate the function of the targeted molecule. For example,when the targeted molecule is an miRNA or pre-miRNA, the NAT can inhibitgene expression; when the target is an miRNA, the NAT will inhibit themiRNA function and will thus up-regulate expression of the mRNAstargeted by the particular miRNA. Alternatively, when the target is aregion of a pre-mRNA that affects splicing, the NAT can alter the choiceof splice site and thus the mRNA sequence; when the NAT functions as anmiRNA, expression of the targeted mRNA is inhibited. For ease ofexposition the term nucleotide or ribonucleotide is sometimes usedherein in reference to one or more monomeric subunits of anoligonucleotide agent. It will be understood that the term“ribonucleotide” or “nucleotide” can, in the case of a modified RNA ornucleotide surrogate, also refer to a modified nucleotide, or surrogatereplacement moiety at one or more positions.

A NAT oligonucleotide agent is, or includes, a region that is at leastpartially, and in some embodiments fully, complementary to the targetRNA. It is not necessary that there be perfect complementarity betweenthe oligonucleotide agent and the target, but the correspondence must besufficient to enable the oligonucleotide agent, or a cleavage productthereof, to modulate (e.g., inhibit) target gene expression.

The oligonucleotide agent will preferably have one or more of thefollowing properties: (1) it will have a 5′ modification that includesone or more phosphate groups or one or more analogs of a phosphategroup; (2) it will, despite modifications even to a very large number ofbases, specifically base pair and form a duplex structure with ahomologous target RNA of sufficient thermodynamic stability to allowmodulation of the activity of the targeted RNA; and (3) it will, despitemodifications even to a very large number, or all of the nucleosides,still have “RNA-like” properties, i.e., it will possess the overallstructural, chemical and physical properties of an RNA molecule, eventhough not exclusively, or even partly, of ribonucleotide-based content.For example, all of the nucleotide sugars can contain a 2′-fluoro groupin place of 2′-hydroxyl group. This deoxyribonucleotide-containing agentcan still be expected to exhibit RNA-like properties. While not wishingto be bound by theory, the electronegative fluorine prefers an axialorientation when attached to the C2′-position of ribose. This spatialpreference of fluorine can force the sugars to adopt a C_(3′)-endopucker. This is the same puckering mode as observed in RNA molecules andgives rise to the RNA-characteristic A-family-type helix. Further, sincefluorine is a good hydrogen bond acceptor, it can participate in thesame hydrogen bonding interactions with water molecules that are knownto stabilize RNA structures. Generally, it is preferred that a modifiedmoiety at the 2′-sugar position will be able to enter intohydrogen-bonding which is more characteristic of the 2′-OH moiety of aribonucleotide than the 2′-H moiety of a deoxyribonucleotide. Apreferred oligonucleotide agent will: exhibit a C_(3′)-endo pucker inall, or at least about 50, 75, 80, 85, 90, or 95% of its sugars; exhibita C_(3′)-endo pucker in a sufficient amount of its sugars that it cangive rise to the RNA-characteristic A-family-type helix; will generallyhave no more than about 20, 10, 5, 4, 3, 2, or 1 sugar which is not aC_(3′)-endo pucker structure. In certain instances, oligonucleotide willexhibit C_(3′)-endo suger pucker and be modified at the 2′-position.Exemplary modifications include 2′-OH, 2′-O-Me, 2′-O-methoxyethyl,2′-O-aminopropyl, 2′-F, 2′-O—CH₂—CO—NHMe, 2′-O—CH₂—CH₂—O—CH₂—CH₂—N(Me)₂,and LNA. In certain instances, regardless of the nature of themodification, and even though the oligonucleotide agent can containdeoxynucleotides or modified deoxynucleotides, it is preferred that DNAmolecules, or any molecule in which more than 50, 60, or 70% of thenucleotides in the molecule are deoxyribonucleotides, or modifieddeoxyribonucleotides which are deoxy at the 2′ position, are excludedfrom the definition of oligonucleotide agent. Some preferred2′-modifications with of sugar moieties exhibiting C2′-endo sugar puckerinclude 2′-H, 2′-Me, 2′-S-Me, 2′-Ethynyl, and 2′-ara-F. Additional sugarmodifications include L-sugars and 2′-5′-linked sugars.

As used herein, “specifically hybridizable” and “complementary” areterms that are used to indicate a sufficient degree of complementaritysuch that stable and specific binding occurs between a compound of theinvention and a target RNA molecule. This nomenclature also applies toinstances when NAT oligonucleotides agents bind to target RNAs. Specificbinding requires a sufficient lack of complementarity to non-targetsequences under conditions in which specific binding is desired, i.e.,under physiological conditions in the case of in vivo assays ortherapeutic treatment, or in the case of in vitro assays, underconditions in which the assays are performed. It has been shown that asingle mismatch between targeted and non-targeted sequences aresufficient to provide discrimination for siRNA targeting of an mRNA(Brummelkamp et al., Cancer Cell, 2002, 2:243).

In certain instances, a NAT oligonucleotide agent is “sufficientlycomplementary” to a target RNA, such that the oligonucleotide agentinhibits production of protein encoded by the target mRNA. The targetRNA can be a pre-mRNA, mRNA, or miRNA endogenous to the subject. Inanother embodiment, the oligonucleotide agent is “exactly complementary”(excluding the SRMS containing subunit(s)) to a target RNA, e.g., thetarget RNA and the oligonucleotide agent can anneal to form a hybridmade exclusively of Watson-Crick base pairs in the region of exactcomplementarity. A “sufficiently complementary” target RNA can include aregion (e.g., of at least about 7 nucleotides) that is exactlycomplementary to a target RNA. Moreover, in some embodiments, theoligonucleotide agent specifically discriminates a single-nucleotidedifference. In this case, the oligonucleotide agent only down-regulatesgene expression if exact complementary is found in the region thesingle-nucleotide difference.

Oligonucleotide agents discussed include otherwise unmodified RNA andDNA as well as RNA and DNA that have been modified. Examples of modifiedRNA and DNA-include modificiations to improve efficacy and polymers ofnucleoside surrogates. Unmodified RNA refers to a molecule in which thecomponents of the nucleic acid, namely sugars, bases, and phosphatemoieties, are the same or essentially the same as that which occur innature, preferably as occur naturally in the human body. The literaturehas referred to rare or unusual, but naturally occurring, RNAs asmodified RNAs. See Limbach et al. Nucleic Acids Res. 1994, 22,2183-2196. Such rare or unusual RNAs, often termed modified RNAs, aretypically the result of a post-transcriptional modification and arewithin the scope of the term unmodified RNA as used herein. Modified RNAas used herein refers to a molecule in which one or more of thecomponents of the nucleic acid, namely sugars, bases, and phosphatemoieties, are different from that which occur in nature, preferablydifferent from that which occurs in the human body. While they arereferred to as “modified RNAs” they will of course, because of themodification, include molecules that are not, strictly speaking, RNAs.Nucleoside surrogates are molecules in which the ribophosphate backboneis replaced with a non-ribophosphate construct that allows the bases tothe presented in the correct spatial relationship such thathybridization is substantially similar to what is seen with aribophosphate backbone, e.g., non-charged mimics of the ribophosphatebackbone.

Sugar Replacement Monomer Subunits (SRMS)

A nucleotide subunit in which the sugar of the subunit has been soreplaced is referred to herein as a sugar replacement modificationsubunit (SRMS). The SRMS includes two “backbone attachment points”(hydroxyl groups), a “tethering attachment point,” and a ligand, whichis connected indirectly to the SRMS via an intervening tether. The SRMSmay be the 5′- or 3′-terminal subunit of the oligonucleotide agent andlocated adjacent to two or more unmodified or modified ribonucleotides.Alternatively, the SRMS may occupy an internal position located adjacentto one or more unmodified or modified ribonucleotides. More than oneSRMS may be present in an oligonucleotide agent. Preferred positions forinclusion of a SRMS tethered to a moiety (e.g., a lipophilic moiety suchas cholesterol) are at the 3′-terminus, the 5′-terminus, or at aninternal position.

Ligands

A wide variety of entities can be tethered to the oligonucleotide agent.A ligand tethered to an oligonucleotide agent can have a favorableeffect on the agent. For example, the ligand can improve stability,hybridization thermodynamics with a target nucleic acid, targeting to aparticular tissue or cell-type, or cell permeability, e.g., by anendocytosis-dependent or -independent mechanism. Ligands and associatedmodifications can also increase sequence specificity and consequentlydecrease off-site targeting. A tethered ligand can include one or moremodified bases or sugars that can function as intercalators. These arepreferably located in an internal region, such as in a bulge of amiRNA/target duplex. The intercalator can be an aromatic group includingpolycyclic aromatics or heterocyclic aromatic groups. A polycyclicintercalator can have stacking capabilities, and can include systemswith 2, 3, or 4 fused rings. Universal bases can be included on aligand.

In one embodiment, the ligand includes a cleaving group that contributesto target gene inhibition by cleavage of the target nucleic acid. Thecleaving group can be a bleomycin (e.g., bleomycin-A5, bleomycin-A2, orbleomycin-B2), pyrene, phenanthroline (e.g., O-phenanthroline), apolyamine, a tripeptide (e.g., lys-tyr-lys tripeptide), or metal-ionchelating group. The metal-ion chelating group can be an Lu(III) orEU(III) macrocyclic complex, a Zn(II) 2,9-dimethylphenanthrolinederivative, a Cu(II) terpyridine, or acridine, which can promote theselective cleavage of target RNA at the site of the bulge by free metalions such as Lu(III). In some instances, a peptide ligand can betethered to a miRNA to promote cleavage of the target RNA. In certaininstances, the cleavage may occur at the bulge region. For example,1,8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane (cyclam) can beconjugated to a peptide, such as via an amino acid derivative, topromote target RNA cleavage.

A tethered ligand can be an aminoglycoside ligand which can cause anoligonucleotide agent to have improved hybridization properties orimproved sequence specificity. Exemplary aminoglycosides includeglycosylated polylysine, galactosylated polylysine, neomycin B,tobramycin, kanamycin A, and acridine conjugates of aminoglycosides,such as Neo-N-acridine, Neo-S-acridine, Neo-C-acridine,Tobra-N-acridine, and KanaA-N-acridine. Use of an acridine analog canincrease sequence specificity. For example, neomycin B has a highaffinity for RNA as compared to DNA, but low sequence-specificity. Anacridine analog, neo-5-acridine has an increased affinity for the HIVRev-response element (RRE). In some embodiments the guanidine analog(the guanidinoglycoside) of an aminoglycoside ligand is tethered to anoligonucleotide agent. In a guanidinoglycoside, the amine group on theamino acid is exchanged for a guanidine group. Attachment of a guanidineanalog can enhance cell permeability of an oligonucleotide agent. Atethered ligand can be a poly-arginine peptide, peptoid orpeptidomimetic, which can enhance the cellular uptake of anoligonucleotide agent.

Preferred moieties are ligands, which are coupled, preferablycovalently, either directly or indirectly via an intervening tether, tothe SRMS carrier. In preferred embodiments, the ligand is attached tothe carrier via an intervening tether. As discussed above, the ligand ortethered ligand may be present on the SRMS monomer when the SRMS monomeris incorporated into the growing strand. In some embodiments, the ligandmay be incorporated into a “precursor” SRMS after a “precursor” SRMSmonomer has been incorporated into the growing strand. For example, anSRMS monomer having an amino-terminated tether (i.e., having noassociated ligand), or TAP-(CH₂)_(n)NH₂ may be incorporated into agrowing oligonucleotide strand. In a subsequent operation, a ligandhaving an electrophilic group can subsequently be attached to theprecursor SRMS by coupling the electrophilic group of the ligand with aterminal nucleophilic group of the precursor SRMS tether. Representativeelectrophilic groups include pentafluorophenyl esters or an aldehyde.Other electrophilic groups amenable to the present invention can bereadily determined by one of ordinary skill in the art.

Induction of DNA Methylation by siRNA

In addition to the well characterized mechanisms of siRNA-induced genesilencing in the cytoplasm, recent studies indicate that siRNA also actsin the nucleus to cause alterations in patterns of DNA methylation,heterochromatin formation, and programmed DNA elimination thus resultingin gene silencing. For reviews, see N. Agrawal et al. Microbiol. Mol.Biol. Rev. 2003, 67, 657-685; Kent, O. A.; MacMillan, A. M. Org. Biomol.Chem. 2004, 2, 1957-1961; Lippman, Z.; Martienssen, R. Nature 2004, 431,364-370; M. Matzke et al. Biochim. Biophys. Acta. 2004, 1677, 129-141;and Schramke, V.; Allshire, R. Curr. Opin. Genet. Dev. 2004, 14,174-180. This silencing requires components of the RNAi machinery, butthe mechanism is not well understood.

Unlike the rest of the nuclear DNA, heterochromatin remains condensedthroughout the cell cycle. Heterochromatin is of interest because of itsability to influence the regulation of nearby genes. Heterochromaticrepeats are not similar in sequence between species, but in all species,heterochromatic DNA is not transcribed, but instead is silenced byconserved epigenetic modifications of histones and DNA itself. Thissilencing is believed to prevent illegitimate recombination. The role ofDNA methylation in silencing has long been recognized. As almost all DNAmethylation is confined to transposons and repeat elements, theseregions must somehow be distinguished from genes. RNAi appears to be onemechanism that allows sequence-specific targeting of methylation.

The first indication that there is a link between the RNAi machinery andheterochromatin formation came from a study in yeast that showed thatdeletion of RNAi associated proteins relieved silencing of genesinserted into centromeric heterochromatin. See T. A. Volpe et al.Science. 2002, 297, 1833-1837. Subsequently, Schramke and Allshiredemonstrated in fission yeast that expression of a synthetic shorthairpin RNA could silence expression of a euchromatic gene. SeeSchramke, V.; Allshire, R. Science 2003, 301, 1069-1074. Silencing wascoupled to chromatin modification and recruitment of heterochromatinproteins and cohesin to the target locus. Silencing via this mechanismrequires Argonaute, Dicer, and RNA-directed RNA polymerase, the knowncomponents of the RNAi machinery. See Volpe et al. cited above.

Biochemical purification of chromodomain complexes in fission yeast hasyielded the RITS (RNAi-induced transcriptional gene silencing) complex.See A. Verdel et al. Science 2004, 303, 672-676. RITS recognizes andbinds to specific chromosome regions to initiate heterochromatic genesilencing. Specific sequence recognition is directed by siRNA. RITScontains Ago1, the S. pombe homolog of the Argonaute family of proteins.At least two subunits of the RITS complex, Chp1 and Tas3, specificallyassociate with the heterochromatic DNA regions, which suggests that thecomplex localizes directly to its target DNA. RITS also contains achromodomain protein, Chp1, which is localized throughoutheterochromatic DNA regions and requires the methyltransferase Clr4 andhistone H3-K9 methylation for localization to chromatin. Thus, RITScontains both a subunit (Ago1) that binds to siRNAs and can function inRNA or DNA targeting by sequence-specific pairing interaction and asubunit (Chp1) that associates with specifically modified histones andmay be involved in further stabilizing its association with chromatin.

Two groups have recently demonstrated that siRNAs can induce DNAmethylation and histone H3 methylation in human cells. See Kawasaki, H.;Taira, K. Nature 2004, 431, 211-217 and Morris et al. Science 2004, 305,1289-1292. It has also been shown that Dicer, the nuclease thatprocesses siRNA from precursor, is required for heterochromatinformation in chicken cells. Fukagawa et al. Nat. Cell Biol. 2004, 6,784-791.

Synthesis of Oligonucleotides of the Invention

siRNA compounds of the invention may be prepared using a two-stepprocedure. First, the individual strands of the double-stranded RNAmolecule are prepared separately. Then, the component strands areannealed. The individual strands of the siRNA compound can be preparedusing solution-phase or solid-phase organic synthesis or both. Organicsynthesis offers the advantage that the oligonucleotide strandscomprising unnatural or modified nucleotides can be easily prepared.Single-stranded oligonucleotides of the invention can be prepared usingsolution-phase or solid-phase organic synthesis or both.

Ligand-conjugated oligonucleotides of the invention may be synthesizedby the use of an oligonucleotide that bears a pendant reactivefunctionality, such as that derived from the attachment of a linkingmolecule onto the oligonucleotide. This reactive oligonucleotide may bereacted directly with commercially-available ligands, ligands that aresynthesized bearing any of a variety of protecting groups, or ligandsthat have a linking moiety attached thereto. The methods of the presentinvention facilitate the synthesis of ligand-conjugated oligonucleotidesby the use of, in some preferred embodiments, nucleoside monomers thathave been appropriately conjugated with ligands and that may further beattached to a solid-support material. Such ligand-nucleoside conjugates,optionally attached to a solid-support material, are prepared accordingto some preferred embodiments of the methods of the present inventionvia reaction of a selected serum-binding ligand with a linking moietylocated on the 5′ position of a nucleoside or oligonucleotide. Incertain instances, an oligonucleotide bearing an aralkyl ligand attachedto the 3′-terminus of the oligonucleotide is prepared by firstcovalently attaching a monomer building block to a controlled-pore-glasssupport via a long-chain aminoalkyl group. Then, nucleotides are bondedvia standard solid-phase synthesis techniques to the monomerbuilding-block bound to the solid support. The monomer building blockmay be a nucleoside or other organic compound that is compatible withsolid-phase synthesis.

The oligonucleotides used in the conjugates of the present invention maybe conveniently and routinely made through the well-known technique ofsolid-phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is also known to usesimilar techniques to prepare other oligonucleotides, such as thephosphorothioates and alkylated derivatives.

Teachings regarding the synthesis of particular modifiedoligonucleotides may be found in the following U.S. patents: U.S. Pat.Nos. 5,138,045 and 5,218,105, drawn to polyamine conjugatedoligonucleotides; U.S. Pat. No. 5,212,295, drawn to monomers for thepreparation of oligonucleotides having chiral phosphorus linkages; U.S.Pat. Nos. 5,378,825 and 5,541,307, drawn to oligonucleotides havingmodified backbones; U.S. Pat. No. 5,386,023, drawn to backbone-modifiedoligonucleotides and the preparation thereof through reductive coupling;U.S. Pat. No. 5,457,191, drawn to modified nucleobases based on the3-deazapurine ring system and methods of synthesis thereof; U.S. Pat.No. 5,459,255, drawn to modified nucleobases based on N-2 substitutedpurines; U.S. Pat. No. 5,521,302, drawn to processes for preparingoligonucleotides having chiral phosphorus linkages; U.S. Pat. No.5,539,082, drawn to peptide nucleic acids; U.S. Pat. No. 5,554,746,drawn to oligonucleotides having β-lactam backbones; U.S. Pat. No.5,571,902, drawn to methods and materials for the synthesis ofoligonucleotides; U.S. Pat. No. 5,578,718, drawn to nucleosides havingalkylthio groups, wherein such groups may be used as linkers to othermoieties attached at any of a variety of positions of the nucleoside;U.S. Pat. Nos. 5,587,361 and 5,599,797, drawn to oligonucleotides havingphosphorothioate linkages of high chiral purity; U.S. Pat. No.5,506,351, drawn to processes for the preparation of 2′-O-alkylguanosine and related compounds, including 2,6-diaminopurine compounds;U.S. Pat. No. 5,587,469, drawn to oligonucleotides having N-2substituted purines; U.S. Pat. No. 5,587,470, drawn to oligonucleotideshaving 3-deazapurines; U.S. Pat. No. 5,223,168, and U.S. Pat. No.5,608,046, both drawn to conjugated 4′-desmethyl nucleoside analogs;U.S. Pat. Nos. 5,602,240, and 5,610,289, drawn to backbone-modifiedoligonucleotide analogs; U.S. Pat. Nos. 6,262,241, and 5,459,255, drawnto, inter alia, methods of synthesizing 2′-fluoro-oligonucleotides.

In the ligand-conjugated oligonucleotides and ligand-molecule bearingsequence-specific linked nucleosides of the present invention, theoligonucleotides and oligonucleosides, may be assembled on a suitableDNA synthesizer utilizing standard nucleotide or nucleoside precursors,or nucleotide or nucleoside conjugate precursors that already bear thelinking moiety, ligand-nucleotide or nucleoside-conjugate precursorsthat already bear the ligand molecule, or non-nucleoside ligand-bearingbuilding blocks.

When using nucleotide-conjugate precursors that already bear a linkingmoiety, the synthesis of the sequence-specific linked nucleosides istypically completed, and the ligand molecule is then reacted with thelinking moiety to form the ligand-conjugated oligonucleotide.Oligonucleotide conjugates bearing a variety of molecules such assteroids, vitamins, lipids and reporter molecules, has previously beendescribed (see Manoharan et al., PCT Application WO 93/07883). In apreferred embodiment, the oligonucleotides or linked nucleosides of thepresent invention are synthesized by an automated synthesizer usingphosphoramidites derived from ligand-nucleoside conjugates in additionto the standard phosphoramidites and non-standard phosphoramidites thatare commercially available and routinely used in oligonucleotidesynthesis.

Incorporation of a 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl, 2′-O-allyl,2′-O-aminoalkyl or 2′-deoxy-2′-fluoro group in nucleosides of anoligonucleotide confers enhanced hybridization properties to theoligonucleotide. Further, oligonucleotides containing phosphorothioatebackbones have enhanced nuclease stability. Thus, functionalized, linkednucleosides of the invention can be augmented to include either or botha phosphorothioate backbone or a 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl,2′-O-aminoalkyl, 2′-O-allyl, or 2′-deoxy-2′-fluoro group.

In some preferred embodiments, functionalized nucleoside sequences ofthe invention possessing an amino group at the 5′-terminus are preparedusing a DNA synthesizer, and then reacted with an active esterderivative of a selected ligand. Active ester derivatives are well knownto those skilled in the art. Representative active esters includeN-hydrosuccinimide esters, tetrafluorophenolic esters,pentafluorophenolic esters and pentachlorophenolic esters. The reactionof the amino group and the active ester produces an oligonucleotide inwhich the selected ligand is attached to the 5′-position through alinking group. The amino group at the 5′-terminus can be preparedutilizing a 5′-Amino-Modifier C6 reagent. In a preferred embodiment,ligand molecules may be conjugated to oligonucleotides at the5′-position by the use of a ligand-nucleoside phosphoramidite whereinthe ligand is linked to the 5′-hydroxy group directly or indirectly viaa linker. Such ligand-nucleoside phosphoramidites are typically used atthe end of an automated synthesis procedure to provide aligand-conjugated oligonucleotide bearing the ligand at the 5′-terminus.

In one preferred embodiment of the methods of the invention, thepreparation of ligand conjugated oligonucleotides commences with theselection of appropriate precursor molecules upon which to construct theligand molecule. Typically, the precursor is an appropriately-protectedderivative of the commonly-used nucleosides. For example, the syntheticprecursors for the synthesis of the ligand-conjugated oligonucleotidesof the present invention include, but are not limited to,2′-aminoalkoxy-5′-ODMT-nucleosides,2′-6-aminoalkylamino-5′-ODMT-nucleosides,5′-6-aminoalkoxy-2′-deoxy-nucleosides,5′-6-aminoalkoxy-2-protected-nucleosides,3′-6-aminoalkoxy-5′-ODMT-nucleosides, and3′-aminoalkylamino-5′-ODMT-nucleosides that may be protected in thenucleobase portion of the molecule. Methods for the synthesis of suchamino-linked protected nucleoside precursors are known to those ofordinary skill in the art.

In many cases, protecting groups are used during the preparation of thecompounds of the invention. As used herein, the term “protected” meansthat the indicated moiety has a protecting group appended thereon. Insome preferred embodiments of the invention, compounds contain one ormore protecting groups. A wide variety of protecting groups can beemployed in the methods of the invention. In general, protecting groupsrender chemical functionalities inert to specific reaction conditions,and can be appended to and removed from such functionalities in amolecule without substantially damaging the remainder of the molecule.

Representative hydroxyl protecting groups, for example, are disclosed byBeaucage et al. (Tetrahedron, 1992, 48:2223-2311). Further hydroxylprotecting groups, as well as other representative protecting groups,are disclosed in Greene and Wuts, Protective Groups in OrganicSynthesis, Chapter 2, 2d ed., John Wiley & Sons, New York, 1991, andOligonucleotides And Analogues A Practical Approach, Ekstein, F. Ed.,IRL Press, N.Y, 1991.

Examples of hydroxyl protecting groups include, but are not limited to,t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl,p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl,diphenylmethyl, p,p′-dinitrobenzhydryl, p-nitrobenzyl, triphenylmethyl,trimethylsilyl, triethylsilyl, t-butyldimethylsilyl,t-butyldiphenylsilyl, triphenylsilyl, benzoylformate, acetate,chloroacetate, trichloroacetate, trifluoroacetate, pivaloate, benzoate,p-phenylbenzoate, 9-fluorenylmethyl carbonate, mesylate and tosylate.

Amino-protecting groups stable to acid treatment are selectively removedwith base treatment, and are used to make reactive amino groupsselectively available for substitution. Examples of such groups are theFmoc (E. Atherton and R. C. Sheppard in The Peptides, S. Udenfriend, J.Meienhofer, Eds., Academic Press, Orlando, 1987, volume 9, p. 1) andvarious substituted sulfonylethyl carbamates exemplified by the Nscgroup (Samukov et al., Tetrahedron Lett., 1994, 35:7821; Verhart andTesser, Rec. Trav. Chim. Pays-Bas, 1987, 107:621).

Additional amino-protecting groups include, but are not limited to,carbamate protecting groups, such as 2-trimethylsilylethoxycarbonyl(Teoc), 1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl(BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl (Fmoc),and benzyloxycarbonyl (Cbz); amide protecting groups, such as formyl,acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl; sulfonamideprotecting groups, such as 2-nitrobenzenesulfonyl; and imine and cyclicimide protecting groups, such as phthalimido and dithiasuccinoyl.Equivalents of these amino-protecting groups are also encompassed by thecompounds and methods of the present invention.

Many solid supports are commercially available and one of ordinary skillin the art can readily select a solid support to be used in thesolid-phase synthesis steps. In certain embodiments, a universal supportis used. A universal support allows for preparation of oligonucleotideshaving unusual or modified nucleotides located at the 3′-terminus of theoligonucleotide. Universal Support 500 and Universal Support II areuniversal supports that are commercially available from Glen Research,22825 Davis Drive, Sterling, Va. For further details about universalsupports see Scott et al., Innovations and Perspectives in solid-phaseSynthesis, 3rd International Symposium, 1994, Ed. Roger Epton, MayflowerWorldwide, 115-124]; Azhayev, A. V. Tetrahedron 1999, 55, 787-800; andAzhayev and Antopolsky Tetrahedron 2001, 57, 4977-4986. In addition, ithas been reported that the oligonucleotide can be cleaved from theuniversal support under milder reaction conditions when oligonucleotideis bonded to the solid support via a syn-1,2-acetoxyphosphate groupwhich more readily undergoes basic hydrolysis. See Guzaev, A. I.;Manoharan, M. J. Am. Chem. Soc. 2003, 125, 2380.

In certain instances, the ribose sugar moiety that naturally occurs innucleosides is replaced with a hexose sugar, polycyclic heteroalkylring, or cyclohexenyl group. In certain instances, the hexose sugar isan allose, altrose, glucose, mannose, gulose, idose, galactose, talose,or a derivative thereof. In a preferred embodiment, the hexose is aD-hexose. In a preferred embodiment, the hexose sugar is glucose ormannose. In certain instances, the polycyclic heteroalkyl group is abicyclic ring containing one oxygen atom in the ring. In certaininstances, the polycyclic heteroalkyl group is a bicyclo[2.2.1]heptane,a bicyclo[3.2.1]octane, or a bicyclo[3.3.1]nonane. In certain instances,the sugar moiety is represented by A′ or A″, and the definition of A²,Y, R⁵, and x is consistent with that described below for theoligonucleotide of formula II.

In certain instances, the sugar moiety is replaced with a non-naturalsugar selected from the group consisting of

R¹ represents independently for each occurrence H, alkyl, or halogen;

R⁵ represents independently for each occurrence H, or an instance of R⁵and R¹² taken together form a 4-, 5-, 6-, 7-, or 8-membered ring; or aninstance of R⁵ and R⁶ taken together form a bond;

R⁶ represents independently for each occurrence H, OH, F, —Oalkyl,—Oallyl, or —Oalkylamine; or an instance of R⁵ and R⁶ taken togetherform a bond; or an instance of R⁶ and R⁸ taken together form a bond;

R⁷, R⁹, and R¹¹ represent independently for each occurrence H, F,—Oalkyl, —Oallyl, or —Oalkylamine;

R⁸ represents independently for each occurrence H, OH, F, —Oalkyl,—Oallyl, or —Oalkylamine; or an instance of R⁶ and R⁸ taken togetherform a bond; or an instance of R⁸ and R¹⁰ taken together form a bond;

R¹⁰ represents independently for each occurrence H, OH, F, —Oalkyl,—Oallyl, or —Oalkylamine; or an instance of R⁸ and R¹⁰ taken togetherform a bond; or an instance of R¹⁰ and R¹² taken together form a bond;

R¹² represents independently for each occurrence for each occurrence H,or an instance of R⁵ and R¹² taken together form a 4-, 5-, 6-, 7-, or8-membered ring; or an instance of R¹⁰ and R¹² taken together form abond;

R²⁵ represents independently for each occurrence H, halogen, alkoxyl,alkyl, aryl, or aralkyl;

R²⁶ represents independently for each occurrence H, halogen, amino,hydroxyl, alkoxyl, alkyl, alkylamino, aryl, aralkyl, —C(O)R²⁷, —CO₂R²⁷,—OC(O)R²⁷—N(R²⁷)COR²⁷, or —N(R²⁷)CO₂R²⁷;

R²⁷ represents independently for each occurrence H, alkyl, aryl, oraralkyl;

w¹ represents independently for each occurrence 0, 1, 2, 3, 4, 5, or 6;

x represents independently for each occurrence 0, 1, 2, or 3; and

the definition Y and A² is the same as presented below foroligonucleotide of formula II.

Therapeutic Uses for Compounds of the Invention

In a preferred embodiment of the present invention, the non-phosphatelinkage enhances the pharmacokinetic properties of the oligonucleotidetherapeutic or diagnostic agent. Such improved pharmacokineticproperties include increased binding of the antisense compound to serumproteins, increased plasma concentration of the antisense compound,increased tissue distribution, increased capacity of binding of theantisense compound to serum proteins, and increased half-lives.

The present invention provides a method for increasing the concentrationof an oligonucleotide in serum. According to such methods, anoligonucleotide comprising a non-phosphate linkage is synthesized andthen added to the serum.

The present invention further provides methods for increasing thecapacity of serum for an oligonucleotide. According to such methods, anoligonucleotide comprising a non-phosphate linkage is synthesized andthen added to the serum.

The present invention also provides methods for increasing the bindingof an oligonucleotide to a portion of the vascular system. According tosuch methods, a vascular protein is selected which resides, in part, inthe circulating serum and, in part, in the non-circulating portion ofthe vascular system. Then, an oligonucleotide comprising a non-phosphatelinkage is synthesized, which is then added to the vascular system. Incertain instances, the oligonucleotide may be conjugated to a ligand toincrease the binding of the oligonucleotide to a portion of the vascularsystem.

The present invention further provides methods for promoting thecellular uptake of an oligonucleotide in a cell. According to suchmethods, a cellular protein is selected. This cellular protein is aprotein that resides on the cellular membrane and extends, in part,extracellularly so that part of this cellular protein extends onto theexternal side of the cellular membrane. Next, an oligonucleotidecomprising a non-phosphate linkage is synthesized and is then broughtinto contact with cells in which cellular uptake of the oligonucleotideis to be promoted.

The present invention also provides methods of increasing cellularuptake of an oligonucleotide comprising contacting an organism with anoligonucleotide of the invention, said oligonucleotide comprising anon-phosphate linkage.

In one preferred embodiment of the invention the protein targeted by theoligonucleotide is a serum protein. It is preferred that the serumprotein targeted by the oligonucleotide compound is an immunoglobulin(an antibody). Preferred immunoglobulins are immunoglobulin G andimmunoglobulin M. Immunoglobulins are known to appear in blood serum andtissues of vertebrate animals.

In another embodiment of the invention the serum protein targeted by theoligonucleotide is a lipoprotein. Lipoproteins are blood proteins havingmolecular weights generally above 20,000 that carry lipids and arerecognized by specific cell-surface receptors. The association withlipoproteins in the serum will initially increase pharmacokineticparameters such as half-life and distribution. A secondary considerationis the ability of lipoproteins to enhance cellular uptake viareceptor-mediated endocytosis.

In yet another embodiment the serum protein targeted by theoligonucleotide compound is α-2-macroglobulin. In yet a furtherembodiment the serum protein targeted by an oligonucleotide compound isα-1-glycoprotein.

Genes and Diseases

One aspect of the invention relates to a method of treating a subject atrisk for or afflicted with unwanted cell proliferation, e.g., malignantor nonmalignant cell proliferation. The method comprises providing anoligonucleotide agent comprising a non-phosphate linkage, wherein theoligonucleotide is homologous to and can silence, e.g., by cleavage, agene which promotes unwanted cell proliferation; and administering atherapeutically effective dose of the oligonucleotide agent to asubject, preferably a human subject.

In a preferred embodiment the gene is a growth factor or growth factorreceptor gene, a kinase, e.g., a protein tyrosine, serine or threoninekinase gene, an adaptor protein gene, a gene encoding a G proteinsuperfamily molecule, or a gene encoding a transcription factor.

In a preferred embodiment the oligonucleotide agent silences the PDGFbeta gene, and thus can be used to treat a subject having or at risk fora disorder characterized by unwanted PDGF beta expression, e.g.,testicular and lung cancers.

In another preferred embodiment the oligonucleotide agent silences theErb-B gene, and thus can be used to treat a subject having or at riskfor a disorder characterized by unwanted Erb-B expression, e.g., breastcancer.

In a preferred embodiment the oligonucleotide agent silences the Srcgene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted Src expression, e.g., colon cancers.

In a preferred embodiment the oligonucleotide agent silences the CRKgene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted CRK expression, e.g., colon and lungcancers.

In a preferred embodiment the oligonucleotide agent silences the GRB2gene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted GRB2 expression, e.g., squamous cellcarcinoma.

In another preferred embodiment the oligonucleotide agent silences theRAS gene, and thus can be used to treat a subject having or at risk fora disorder characterized by unwanted RAS expression, e.g., pancreatic,colon and lung cancers, and chronic leukemia.

In another preferred embodiment the oligonucleotide agent silences theMEKK gene, and thus can be used to treat a subject having or at risk fora disorder characterized by unwanted MEKK expression, e.g., squamouscell carcinoma, melanoma or leukemia.

In another preferred embodiment the oligonucleotide agent silences theJNK gene, and thus can be used to treat a subject having or at risk fora disorder characterized by unwanted JNK expression, e.g., pancreatic orbreast cancers.

In a preferred embodiment the oligonucleotide agent silences the RAFgene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted RAF expression, e.g., lung cancer orleukemia.

In a preferred embodiment the oligonucleotide agent silences the Erk1/2gene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted Erk1/2 expression, e.g., lung cancer.

In another preferred embodiment the oligonucleotide agent silences thePCNA(p21) gene, and thus can be used to treat a subject having or atrisk for a disorder characterized by unwanted PCNA expression, e.g.,lung cancer.

In a preferred embodiment the oligonucleotide agent silences the MYBgene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted MYB expression, e.g., colon cancer orchronic myelogenous leukemia.

In a preferred embodiment the oligonucleotide agent silences the c-MYCgene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted c-MYC expression, e.g., Burkitt'slymphoma or neuroblastoma.

In another preferred embodiment the oligonucleotide agent silences theJUN gene, and thus can be used to treat a subject having or at risk fora disorder characterized by unwanted JUN expression, e.g., ovarian,prostate or breast cancers.

In another preferred embodiment the oligonucleotide agent silences theFOS gene, and thus can be used to treat a subject having or at risk fora disorder characterized by unwanted FOS expression, e.g., skin orprostate cancers.

In a preferred embodiment the oligonucleotide agent silences the BCL-2gene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted BCL-2 expression, e.g., lung orprostate cancers or Non-Hodgkin lymphoma.

In a preferred embodiment the oligonucleotide agent silences the CyclinD gene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted Cyclin D expression, e.g., esophagealand colon cancers.

In a preferred embodiment the oligonucleotide agent silences the VEGFgene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted VEGF expression, e.g., esophageal andcolon cancers.

In a preferred embodiment the oligonucleotide agent silences the EGFRgene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted EGFR expression, e.g., breast cancer.

In another preferred embodiment the oligonucleotide agent silences theCyclin A gene, and thus can be used to treat a subject having or at riskfor a disorder characterized by unwanted Cyclin A expression, e.g., lungand cervical cancers.

In another preferred embodiment the oligonucleotide agent silences theCyclin E gene, and thus can be used to treat a subject having or at riskfor a disorder characterized by unwanted Cyclin E expression, e.g., lungand breast cancers.

In another preferred embodiment the oligonucleotide agent silences theWNT-1 gene, and thus can be used to treat a subject having or at riskfor a disorder characterized by unwanted WNT-1 expression, e.g., basalcell carcinoma.

In another preferred embodiment the oligonucleotide agent silences thebeta-catenin gene, and thus can be used to treat a subject having or atrisk for a disorder characterized by unwanted beta-catenin expression,e.g., adenocarcinoma or hepatocellular carcinoma.

In another preferred embodiment the oligonucleotide agent silences thec-MET gene, and thus can be used to treat a subject having or at riskfor a disorder characterized by unwanted c-MET expression, e.g.,hepatocellular carcinoma.

In another preferred embodiment the oligonucleotide agent silences thePKC gene, and thus can be used to treat a subject having or at risk fora disorder characterized by unwanted PKC expression, e.g., breastcancer.

In a preferred embodiment the oligonucleotide agent silences the NFKBgene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted NFKB expression, e.g., breast cancer.

In a preferred embodiment the oligonucleotide agent silences the STAT3gene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted STAT3 expression, e.g., prostatecancer.

In another preferred embodiment the oligonucleotide agent silences thesurvivin gene, and thus can be used to treat a subject having or at riskfor a disorder characterized by unwanted survivin expression, e.g.,cervical or pancreatic cancers.

In another preferred embodiment the oligonucleotide agent silences theHer2/Neu gene, and thus can be used to treat a subject having or at riskfor a disorder characterized by unwanted Her2/Neu expression, e.g.,breast cancer.

In another preferred embodiment the oligonucleotide agent silences thetopoisomerase I gene, and thus can be used to treat a subject having orat risk for a disorder characterized by unwanted topoisomerase Iexpression, e.g., ovarian and colon cancers.

In a preferred embodiment the oligonucleotide agent silences thetopoisomerase II alpha gene, and thus can be used to treat a subjecthaving or at risk for a disorder characterized by unwanted topoisomeraseII expression, e.g., breast and colon cancers.

In a preferred embodiment the oligonucleotide agent silences mutationsin the p73 gene, and thus can be used to treat a subject having or atrisk for a disorder characterized by unwanted p73 expression, e.g.,colorectal adenocarcinoma.

In a preferred embodiment the oligonucleotide agent silences mutationsin the p21(WAF1/CIP1) gene, and thus can be used to treat a subjecthaving or at risk for a disorder characterized by unwantedp21(WAF1/CIP1) expression, e.g., liver cancer.

In a preferred embodiment the oligonucleotide agent silences mutationsin the p27(KIP1) gene, and thus can be used to treat a subject having orat risk for a disorder characterized by unwanted p27(KIP1) expression,e.g., liver cancer.

In a preferred embodiment the oligonucleotide agent silences mutationsin the PPM1D gene, and thus can be used to treat a subject having or atrisk for a disorder characterized by unwanted PPM1D expression, e.g.,breast cancer.

In a preferred embodiment the oligonucleotide agent silences mutationsin the RAS gene, and thus can be used to treat a subject having or atrisk for a disorder characterized by unwanted RAS expression, e.g.,breast cancer.

In another preferred embodiment the oligonucleotide agent silencesmutations in the caveolin I gene, and thus can be used to treat asubject having or at risk for a disorder characterized by unwantedcaveolin I expression, e.g., esophageal squamous cell carcinoma.

In another preferred embodiment the oligonucleotide agent silencesmutations in the MIB I gene, and thus can be used to treat a subjecthaving or at risk for a disorder characterized by unwanted MIB Iexpression, e.g., male breast carcinoma (MBC).

In another preferred embodiment the oligonucleotide agent silencesmutations in the MTAI gene, and thus can be used to treat a subjecthaving or at risk for a disorder characterized by unwanted MTAIexpression, e.g., ovarian carcinoma.

In another preferred embodiment the oligonucleotide agent silencesmutations in the M68 gene, and thus can be used to treat a subjecthaving or at risk for a disorder characterized by unwanted M68expression, e.g., human adenocarcinomas of the esophagus, stomach,colon, and rectum.

In preferred embodiments the oligonucleotide agent silences mutations intumor suppressor genes, and thus can be used as a method to promoteapoptotic activity in combination with chemotherapeutics.

In a preferred embodiment the oligonucleotide agent silences mutationsin the p53 tumor suppressor gene, and thus can be used to treat asubject having or at risk for a disorder characterized by unwanted p53expression, e.g., gall bladder, pancreatic and lung cancers.

In a preferred embodiment the oligonucleotide agent silences mutationsin the p53 family member DN-p63, and thus can be used to treat a subjecthaving or at risk for a disorder characterized by unwanted DN-p63expression, e.g., squamous cell carcinoma

In a preferred embodiment the oligonucleotide agent silences mutationsin the pRb tumor suppressor gene, and thus can be used to treat asubject having or at risk for a disorder characterized by unwanted pRbexpression, e.g., oral squamous cell carcinoma

In a preferred embodiment the oligonucleotide agent silences mutationsin the APC1 tumor suppressor gene, and thus can be used to treat asubject having or at risk for a disorder characterized by unwanted APC1expression, e.g., colon cancer.

In a preferred embodiment the oligonucleotide agent silences mutationsin the BRCA1 tumor suppressor gene, and thus can be used to treat asubject having or at risk for a disorder characterized by unwanted BRCA1expression, e.g., breast cancer.

In a preferred embodiment the oligonucleotide agent silences mutationsin the PTEN tumor suppressor gene, and thus can be used to treat asubject having or at risk for a disorder characterized by unwanted PTENexpression, e.g., hamartomas, gliomas, and prostate and endometrialcancers.

In a preferred embodiment the oligonucleotide agent silences mLL fusiongenes, e.g., mLL-AF9, and thus can be used to treat a subject having orat risk for a disorder characterized by unwanted mLL fusion geneexpression, e.g., acute leukemias.

In another preferred embodiment the oligonucleotide agent silences theBCR/ABL fusion gene, and thus can be used to treat a subject having orat risk for a disorder characterized by unwanted BCR/ABL fusion geneexpression, e.g., acute and chronic leukemias.

In another preferred embodiment the oligonucleotide agent silences theTEL/AML1 fusion gene, and thus can be used to treat a subject having orat risk for a disorder characterized by unwanted TEL/AML1 fusion geneexpression, e.g., childhood acute leukemia.

In another preferred embodiment the oligonucleotide agent silences theEWS/FLI1 fusion gene, and thus can be used to treat a subject having orat risk for a disorder characterized by unwanted EWS/FLI1 fusion geneexpression, e.g., Ewing Sarcoma.

In another preferred embodiment the oligonucleotide agent silences theTLS/FUS1 fusion gene, and thus can be used to treat a subject having orat risk for a disorder characterized by unwanted TLS/FUS1 fusion geneexpression, e.g., Myxoid liposarcoma.

In another preferred embodiment the oligonucleotide agent silences thePAX3/FKHR fusion gene, and thus can be used to treat a subject having orat risk for a disorder characterized by unwanted PAX3/FKHR fusion geneexpression, e.g., Myxoid liposarcoma.

In another preferred embodiment the oligonucleotide agent silences theAML1/ETO fusion gene, and thus can be used to treat a subject having orat risk for a disorder characterized by unwanted AML1/ETO fusion geneexpression, e.g., acute leukemia.

Another aspect of the invention relates to a method of treating asubject, e.g., a human, at risk for or afflicted with a disease ordisorder that may benefit by angiogenesis inhibition e.g., cancer. Themethod comprises providing an oligonucleotide agent comprising anon-phosphate linkage, wherein said oligonucleotide agent is homologousto and can silence, e.g., by cleavage, a gene which mediatesangiogenesis; and administering a therapeutically effective dosage ofsaid oligonucleotide agent to a subject, preferrably a human.

In a preferred embodiment the oligonucleotide agent silences the alphav-integrin gene, and thus can be used to treat a subject having or atrisk for a disorder characterized by unwanted alpha V integrin, e.g.,brain tumors or tumors of epithelial origin.

In a preferred embodiment the oligonucleotide agent silences the Flt-1receptor gene, and thus can be used to treat a subject having or at riskfor a disorder characterized by unwanted Flt-1 receptors, eg. cancer andrheumatoid arthritis.

In a preferred embodiment the oligonucleotide agent silences the tubulingene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted tubulin, eg. cancer and retinalneovascularization.

In a preferred embodiment the oligonucleotide agent silences the tubulingene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted tubulin, eg. cancer and retinalneovascularization.

Another aspect of the invention relates to a method of treating asubject infected with a virus or at risk for or afflicted with adisorder or disease associated with a viral infection. The methodcomprises providing an oligonucleotide agent comprising a non-phosphatelinkage, wherein said oligonucleotide agent is homologous to and cansilence, e.g., by cleavage, a viral gene of a cellular gene whichmediates viral function, e.g., entry or growth; and administering atherapeutically effective dose of said oligonucleotide agent to asubject, preferably a human subject.

Thus, the invention provides for a method of treating patients infectedby the Human Papilloma Virus (HPV) or at risk for or afflicted with adisorder mediated by HPV, e.g, cervical cancer. HPV is linked to 95% ofcervical carcinomas and thus an antiviral therapy is an attractivemethod to treat these cancers and other symptoms of viral infection.

In a preferred embodiment, the expression of a HPV gene is reduced. Inanother preferred embodiment, the HPV gene is one of the group of E2,E6, or E7.

In a preferred embodiment the expression of a human gene that isrequired for HPV replication is reduced.

The invention also includes a method of treating patients infected bythe Human Immunodeficiency Virus (HIV) or at risk for or afflicted witha disorder mediated by HIV, e.g., Acquired Immune Deficiency Syndrome(AIDS). In a preferred embodiment, the expression of a HIV gene isreduced. In another preferred embodiment, the HIV gene is CCR5, Gag, orRev. In a preferred embodiment the expression of a human gene that isrequired for HIV replication is reduced. In another preferredembodiment, the gene is CD4 or Tsg101.

The invention also includes a method for treating patients infected bythe Hepatitis B Virus (HBV) or at risk for or afflicted with a disordermediated by HBV, e.g., cirrhosis and heptocellular carcinoma. In apreferred embodiment, the expression of a HBV gene is reduced. Inanother preferred embodiment, the targeted HBV gene encodes one of thegroup of the tail region of the HBV core protein, the pre-cregious(pre-c) region, or the cregious (c) region. In another preferredembodiment, a targeted HBV-RNA sequence is comprised of the poly(A)tail.

In preferred embodiment the expression of a human gene that is requiredfor HBV replication is reduced.

The invention also provides for a method of treating patients infectedby the Hepatitis A Virus (HAV), or at risk for or afflicted with adisorder mediated by HAV. In a preferred embodiment the expression of ahuman gene that is required for HAV replication is reduced.

The present invention provides for a method of treating patientsinfected by the Hepatitis C Virus (HCV), or at risk for or afflictedwith a disorder mediated by HCV, e.g., cirrhosis. In a preferredembodiment, the expression of a HCV gene is reduced. In anotherpreferred embodiment the expression of a human gene that is required forHCV replication is reduced.

The present invention also provides for a method of treating patientsinfected by the any of the group of Hepatitis Viral strains comprisinghepatitis D, E, F, G, or H, or patients at risk for or afflicted with adisorder mediated by any of these strains of hepatitis. In a preferredembodiment, the expression of a Hepatitis, D, E, F, G, or H gene isreduced. In another preferred embodiment the expression of a human genethat is required for hepatitis D, E, F, G or H replication is reduced.

Methods of the invention also provide for treating patients infected bythe Respiratory Syncytial Virus (RSV) or at risk for or afflicted with adisorder mediated by RSV, e.g, lower respiratory tract infection ininfants and childhood asthma, pneumonia and other complications, e.g.,in the elderly. In a preferred embodiment, the expression of a RSV geneis reduced. In another preferred embodiment, the targeted HBV geneencodes one of the group of genes N, L, or P. In a preferred embodimentthe expression of a human gene that is required for RSV replication isreduced.

Methods of the invention provide for treating patients infected by theHerpes Simplex Virus (HSV) or at risk for or afflicted with a disordermediated by HSV, e.g, genital herpes and cold sores as well aslife-threatening or sight-impairing disease mainly in immunocompromisedpatients. In a preferred embodiment, the expression of a HSV gene isreduced. In another preferred embodiment, the targeted HSV gene encodesDNA polymerase or the helicase-primase. In a preferred embodiment theexpression of a human gene that is required for HSV replication isreduced.

The invention also provides a method for treating patients infected bythe herpes Cytomegalovirus (CMV) or at risk for or afflicted with adisorder mediated by CMV, e.g., congenital virus infections andmorbidity in immunocompromised patients. In a preferred embodiment, theexpression of a CMV gene is reduced. In a preferred embodiment theexpression of a human gene that is required for CMV replication isreduced.

Methods of the invention also provide for a method of treating patientsinfected by the herpes Epstein Barr Virus (EBV) or at risk for orafflicted with a disorder mediated by EBV, e.g., NK/T-cell lymphoma,non-Hodgkin lymphoma, and Hodgkin disease. In a preferred embodiment,the expression of a EBV gene is reduced. In a preferred embodiment theexpression of a human gene that is required for EBV replication isreduced.

Methods of the invention also provide for treating patients infected byKaposi's Sarcoma-associated Herpes Virus (KSHV), also called humanherpesvirus 8, or patients at risk for or afflicted with a disordermediated by KSHV, e.g., Kaposi's sarcoma, multicentric Castleman'sdisease and AIDS-associated primary effusion lymphoma. In a preferredembodiment, the expression of a KSHV gene is reduced. In a preferredembodiment the expression of a human gene that is required for KSHVreplication is reduced.

The invention also includes a method for treating patients infected bythe JC Virus (JCV) or a disease or disorder associated with this virus,e.g., progressive multifocal leukoencephalopathy (PML). In a preferredembodiment, the expression of a JCV gene is reduced. In preferredembodiment the expression of a human gene that is required for JCVreplication is reduced.

Methods of the invention also provide for treating patients infected bythe myxovirus or at risk for or afflicted with a disorder mediated bymyxovirus, e.g., influenza. In a preferred embodiment, the expression ofa myxovirus gene is reduced. In a preferred embodiment the expression ofa human gene that is required for myxovirus replication is reduced.

Methods of the invention also provide for treating patients infected bythe rhinovirus or at risk for of afflicted with a disorder mediated byrhinovirus, e.g., the common cold. In a preferred embodiment, theexpression of a rhinovirus gene is reduced. In preferred embodiment theexpression of a human gene that is required for rhinovirus replicationis reduced.

Methods of the invention also provide for treating patients infected bythe coronavirus or at risk for of afflicted with a disorder mediated bycoronavirus, e.g., the common cold. In a preferred embodiment, theexpression of a coronavirus gene is reduced. In preferred embodiment theexpression of a human gene that is required for coronavirus replicationis reduced.

Methods of the invention also provide for treating patients infected bythe flavivirus West Nile or at risk for or afflicted with a disordermediated by West Nile Virus. In a preferred embodiment, the expressionof a West Nile Virus gene is reduced. In another preferred embodiment,the West Nile Virus gene is one of the group comprising E, NS3, or NS5.In a preferred embodiment the expression of a human gene that isrequired for West Nile Virus replication is reduced.

Methods of the invention also provide for treating patients infected bythe St. Louis Encephalitis flavivirus, or at risk for or afflicted witha disease or disorder associated with this virus, e.g., viralhaemorrhagic fever or neurological disease. In a preferred embodiment,the expression of a St. Louis Encephalitis gene is reduced. In apreferred embodiment the expression of a human gene that is required forSt. Louis Encephalitis virus replication is reduced.

Methods of the invention also provide for treating patients infected bythe Tick-borne encephalitis flavivirus, or at risk for or afflicted witha disorder mediated by Tick-borne encephalitis virus, e.g., viralhaemorrhagic fever and neurological disease. In a preferred embodiment,the expression of a Tick-borne encephalitis virus gene is reduced. In apreferred embodiment the expression of a human gene that is required forTick-borne encephalitis virus replication is reduced.

Methods of the invention also provide for methods of treating patientsinfected by the Murray Valley encephalitis flavivirus, which commonlyresults in viral haemorrhagic fever and neurological disease. In apreferred embodiment, the expression of a Murray Valley encephalitisvirus gene is reduced. In a preferred embodiment the expression of ahuman gene that is required for Murray Valley encephalitis virusreplication is reduced.

The invention also includes methods for treating patients infected bythe dengue flavivirus, or a disease or disorder associated with thisvirus, e.g., dengue haemorrhagic fever. In a preferred embodiment, theexpression of a dengue virus gene is reduced. In a preferred embodimentthe expression of a human gene that is required for dengue virusreplication is reduced.

Methods of the invention also provide for treating patients infected bythe Simian Virus 40 (SV40) or at risk for or afflicted with a disordermediated by SV40, e.g., tumorigenesis. In a preferred embodiment, theexpression of a SV40 gene is reduced. In a preferred embodiment theexpression of a human gene that is required for SV40 replication isreduced.

The invention also includes methods for treating patients infected bythe Human T Cell Lymphotropic Virus (HTLV), or a disease or disorderassociated with this virus, e.g., leukemia and myelopathy. In apreferred embodiment, the expression of a HTLV gene is reduced. Inanother preferred embodiment the HTLV1 gene is the Tax transcriptionalactivator. In a preferred embodiment the expression of a human gene thatis required for HTLV replication is reduced.

Methods of the invention also provide for treating patients infected bythe Moloney-Murine Leukemia Virus (Mo-MuLV) or at risk for or afflictedwith a disorder mediated by Mo-MuLV, e.g., T-cell leukemia. In apreferred embodiment, the expression of a Mo-MuLV gene is reduced. In apreferred embodiment the expression of a human gene that is required forMo-MuLV replication is reduced.

Methods of the invention also provide for treating patients infected bythe encephalomyocarditis virus (EMCV) or at risk for or afflicted with adisorder mediated by EMCV, e.g. myocarditis. EMCV leads to myocarditisin mice and pigs and is capable of infecting human myocardial cells.This virus is therefore a concern for patients undergoingxenotransplantation. In a preferred embodiment, the expression of a EMCVgene is reduced. In a preferred embodiment the expression of a humangene that is required for EMCV replication is reduced.

The invention also includes a method for treating patients infected bythe measles virus (MV) or at risk for or afflicted with a disordermediated by MV, e.g. measles. In a preferred embodiment, the expressionof a MV gene is reduced. In a preferred embodiment the expression of ahuman gene that is required for MV replication is reduced.

The invention also includes a method for treating patients infected bythe Vericella zoster virus (VZV) or at risk for or afflicted with adisorder mediated by VZV, e.g. chicken pox or shingles (also calledzoster). In a preferred embodiment, the expression of a VZV gene isreduced. In a preferred embodiment the expression of a human gene thatis required for VZV replication is reduced.

The invention also includes a method for treating patients infected byan adenovirus or at risk for or afflicted with a disorder mediated by anadenovirus, e.g. respiratory tract infection. In a preferred embodiment,the expression of an adenovirus gene is reduced. In a preferredembodiment the expression of a human gene that is required foradenovirus replication is reduced.

The invention includes a method for treating patients infected by ayellow fever virus (YFV) or at risk for or afflicted with a disordermediated by a YFV, e.g. respiratory tract infection. In a preferredembodiment, the expression of a YFV gene is reduced. In anotherpreferred embodiment, the preferred gene is one of a group that includesthe E, NS2A, or NS3 genes. In a preferred embodiment the expression of ahuman gene that is required for YFV replication is reduced.

Methods of the invention also provide for treating patients infected bythe poliovirus or at risk for or afflicted with a disorder mediated bypoliovirus, e.g., polio. In a preferred embodiment, the expression of apoliovirus gene is reduced. In a preferred embodiment the expression ofa human gene that is required for poliovirus replication is reduced.

Methods of the invention also provide for treating patients infected bya poxvirus or at risk for or afflicted with a disorder mediated by apoxvirus, e.g., smallpox. In a preferred embodiment, the expression of apoxvirus gene is reduced. In a preferred embodiment the expression of ahuman gene that is required for poxvirus replication is reduced.

In another, aspect the invention features methods of treating a subjectinfected with a pathogen, e.g., a bacterial, amoebic, parasitic, orfungal pathogen. The method comprises providing an oligonucleotide agentcomprising a non-phosphate linkage, wherein said oligonucleotide ishomologous to and can silence, e.g., by cleavage of a pathogen gene; andadministering a therapeutically effective dose of said oligonucleotideagent to a subject, prefereably a human subject.

The target gene can be one involved in growth, cell wall synthesis,protein synthesis, transcription, energy metabolism, e.g., the Krebscycle, or toxin production. Thus, the present invention provides for amethod of treating patients infected by a plasmodium that causesmalaria. In a preferred embodiment, the expression of a plasmodium geneis reduced. In another preferred embodiment, the gene is apical membraneantigen 1 (AMA1). In a preferred embodiment the expression of a humangene that is required for plasmodium replication is reduced.

The invention also includes methods for treating patients infected bythe Mycobacterium ulcerans, or a disease or disorder associated withthis pathogen, e.g., Buruli ulcers. In a preferred embodiment, theexpression of a Mycobacterium ulcerans gene is reduced. In a preferredembodiment the expression of a human gene that is required forMycobacterium ulcerans replication is reduced.

The invention also includes methods for treating patients infected bythe Mycobacterium tuberculosis, or a disease or disorder associated withthis pathogen, e.g., tuberculosis. In a preferred embodiment, theexpression of a Mycobacterium tuberculosis gene is reduced. In apreferred embodiment the expression of a human gene that is required forMycobacterium tuberculosis replication is reduced.

The invention also includes methods for treating patients infected bythe Mycobacterium leprae, or a disease or disorder associated with thispathogen, e.g. leprosy. In a preferred embodiment, the expression of aMycobacterium leprae gene is reduced. In a preferred embodiment theexpression of a human gene that is required for Mycobacterium lepraereplication is reduced.

The invention also includes methods for treating patients infected bythe bacteria Staphylococcus aureus, or a disease or disorder associatedwith this pathogen, e.g. infections of the skin and muscous membranes.In a preferred embodiment, the expression of a Staphylococcus aureusgene is reduced. In a preferred embodiment the expression of a humangene that is required for Staphylococcus aureus replication is reduced.

The invention also includes methods for treating patients infected bythe bacteria Streptococcus pneumoniae, or a disease or disorderassociated with this pathogen, e.g. pneumonia or childhood lowerrespiratory tract infection. In a preferred embodiment, the expressionof a Streptococcus pneumoniae gene is reduced. In a preferred embodimentthe expression of a human gene that is required for Streptococcuspneumoniae replication is reduced.

The invention also includes methods for treating patients infected bythe bacteria Streptococcus pyogenes, or a disease or disorder associatedwith this pathogen, e.g. Strep throat or Scarlet fever. In a preferredembodiment, the expression of a Streptococcus pyogenes gene is reduced.In a preferred embodiment the expression of a human gene that isrequired for Streptococcus pyogenes replication is reduced.

The invention also includes methods for treating patients infected bythe bacteria Chlamydia pneumoniae, or a disease or disorder associatedwith this pathogen, e.g. pneumonia or childhood lower respiratory tractinfection. In a preferred embodiment, the expression of a Chlamydiapneumoniae gene is reduced. In a preferred embodiment the expression ofa human gene that is required for Chlamydia pneumoniae replication isreduced.

The invention also includes methods for treating patients infected bythe bacteria Mycoplasma pneumoniae, or a disease or disorder associatedwith this pathogen, e.g. pneumonia or childhood lower respiratory tractinfection. In a preferred embodiment, the expression of a Mycoplasmapneumoniae gene is reduced. In a preferred embodiment the expression ofa human gene that is required for Mycoplasma pneumoniae replication isreduced.

Another aspect of the invention relates to a method of treating asubject, e.g., a human, at risk for or afflicted with a disease ordisorder characterized by an unwanted immune response, e.g., aninflammatory disease or disorder, or an autoimmune disease or disorder.The method comprises providing an oligonucleotide agent comprising an anon-phosphate linkage, wherein said oligonucleotide agent is homologousto and can silence, e.g., by cleavage, a gene which mediates an unwantedimmune response; and administering said oligonucleotide agent to asubject, preferrably a human subject. In a preferred embodiment thedisease or disorder is an ischemia or reperfusion injury, e.g., ischemiaor reperfusion injury associated with acute myocardial infarction,unstable angina, cardiopulmonary bypass, surgical intervention e.g.,angioplasty, e.g., percutaneous transluminal coronary angioplasty, theresponse to a transplantated organ or tissue, e.g., transplanted cardiacor vascular tissue; or thrombolysis. In a preferred embodiment thedisease or disorder is restenosis, e.g., restenosis associated withsurgical intervention e.g., angioplasty, e.g., percutaneous transluminalcoronary angioplasty. In a prefered embodiment the disease or disorderis Inflammatory Bowel Disease, e.g., Crohn Disease or UlcerativeColitis. In a prefered embodiment the disease or disorder isinflammation associated with an infection or injury. In a preferedembodiment the disease or disorder is asthma, lupus, multiple sclerosis,diabetes, e.g., type II diabetes, arthritis, e.g., rheumatoid orpsoriatic. In particularly preferred embodiments the oligonucleotideagent silences an integrin or co-ligand thereof, e.g., VLA4, VCAM, ICAM.In particularly preferred embodiments the oligonucleotide agent silencesa selectin or co-ligand thereof, e.g., P-selectin, E-selectin (ELAM),I-selectin, P-selectin glycoprotein-1 (PSGL-1). In particularlypreferred embodiments the oligonucleotide agent silences a component ofthe complement system, e.g., C3, C5, C3aR, C5aR, C3 convertase, and C5convertase.

In particularly preferred embodiments the oligonucleotide agent silencesa chemokine or receptor thereof, e.g., TNFI, TNFJ, IL-1I, IL-1J, IL-2,IL-2R, IL-4, IL-4R, IL-5, IL-6, IL-8, TNFRI, TNFRII, IgE, SCYA11, andCCR3.

In other embodiments the oligonucleotide agent silences GCSF, Gro1,Gro2, Gro3, PF4, MIG, Pro-Platelet Basic Protein (PPBP), MIP-1I, MIP-1J,RANTES, MCP-1, MCP-2, MCP-3, CMBKR1, CMBKR2, CMBKR3, CMBKR5, AIF-1, orI-309.

Another aspect of the invention features, a method of treating asubject, e.g., a human, at risk for or afflicted with acute pain orchronic pain. The method comprises providing an oligonucleotide agentcomprising a non-phosphate linkage, wherein said oligonucleotide ishomologous to and can silence, e.g., by cleavage, a gene which mediatesthe processing of pain; and administering a therapeutically effectivedose of said oligonucleotide agent to a subject, preferrably a humansubject. In particularly preferred embodiments the oligonucleotide agentsilences a component of an ion channel. In particularly preferredembodiments the oligonucleotide agent silences a neurotransmitterreceptor or ligand.

Another aspect of the invention relates to a method of treating asubject, e.g., a human, at risk for or afflicted with a neurologicaldisease or disorder. The method comprises providing a oligonucleotideagent comprising a non-phosphate linkage, wherein said oligonucleotideis homologous to and can silence, e.g., by cleavage, a gene whichmediates a neurological disease or disorder; and administering atherapeutically effective dose of said oligonucleotide agent the to asubject, preferrably a human. In a prefered embodiment the disease ordisorder is Alzheimer Disease or Parkinson Disease. In particularlypreferred embodiments the oligonucleotide agent silences anamyloid-family gene, e.g., APP; a presenilin gene, e.g., PSEN1 andPSEN2, or I-synuclein. In a preferred embodiment the disease or disorderis a neurodegenerative trinucleotide repeat disorder, e.g., Huntingtondisease, dentatorubral pallidoluysian atrophy or a spinocerebellarataxia, e.g., SCA1, SCA2, SCA3 (Machado-Joseph disease), SCA7 or SCA8.

In particularly preferred embodiments the oligonucleotide agent silencesHD, DRPLA, SCA1, SCA2, MJD1, CACNL1A4, SCA7, or SCA8.

The loss of heterozygosity (LOH) can result in hemizygosity forsequence, e.g., genes, in the area of LOH. This can result in asignificant genetic difference between normal and disease-state cells,e.g., cancer cells, and provides a useful difference between normal anddisease-state cells, e.g., cancer cells. This difference can arisebecause a gene or other sequence is heterozygous in euploid cells but ishemizygous in cells having LOH. The regions of LOH will often include agene, the loss of which promotes unwanted proliferation, e.g., a tumorsuppressor gene, and other sequences including, e.g., other genes, insome cases a gene which is essential for normal function, e.g., growth.Methods of the invention rely, in part, on the specific cleavage orsilencing of one allele of an essential gene with an oligonucleotideagent of the invention. The oligonucleotide agent is selected such thatit targets the single allele of the essential gene found in the cellshaving LOH but does not silence the other allele, which is present incells which do not show LOH. In essence, it discriminates between thetwo alleles, preferentially silencing the selected allele. In essencepolymorphisms, e.g., SNPs of essential genes that are affected by LOH,are used as a target for a disorder characterized by cells having LOH,e.g., cancer cells having LOH. E.g., one of ordinary skill in the artcan identify essential genes which are in proximity to tumor suppressorgenes, and which are within a LOH region which includes the tumorsuppressor gene. The gene encoding the large subunit of human RNApolymerase II, POLR2A, a gene located in close proximity to the tumorsuppressor gene p53, is such a gene. It frequently occurs within aregion of LOH in cancer cells. Other genes that occur within LOH regionsand are lost in many cancer cell types include the group comprisingreplication protein A 70-kDa subunit, replication protein A 32-kD,ribonucleotide reductase, thymidilate synthase, TATA associated factor2H, ribosomal protein S14, eukaryotic initiation factor 5A, alanyl tRNAsynthetase, cysteinyl tRNA synthetase, NaK ATPase, alpha-1 subunit, andtransferrin receptor.

Accordingly, another aspect of the invention relates to a method oftreating a disorder characterized by LOH, e.g., cancer. The methodcomprises optionally, determining the genotype of the allele of a genein the region of LOH and preferably determining the genotype of bothalleles of the gene in a normal cell; providing an oligonucleotide agentcomprising a non-phosphate linkage which preferentially cleaves orsilences the allele found in the LOH cells; and administerning atherapeutically effective dose of said oligonucleotide agent to thesubject, preferrably a human.

The invention also includes an oligonucleotide agent comprising anon-phosphate linkage disclosed herein, e.g, an oligonucleotide agentwhich can preferentially silence, e.g., cleave, one allele of apolymorphic gene.

In another aspect, the invention provides a method of cleaving orsilencing more than one gene with an oligonucleotide agent comprising anon-phosphate linkage. In these embodiments the oligonucleotide agent isselected so that it has sufficient homology to a sequence found in morethan one gene. For example, the sequence AAGCTGGCCCTGGACATGGAGAT isconserved between mouse lamin B1, lamin B2, keratin complex 2-gene 1 andlamin A/C. Thus an oligonucleotide agent targeted to this sequence wouldeffectively silence the entire collection of genes.

The invention also includes an oligonucleotide agent comprising anon-phosphate linkage disclosed herein, which can silence more than onegene.

Compounds of the Invention

One aspect of the present invention relates to 3′-phosphonamiditesubstituted nucleosides represented by formula I:

wherein

R¹ is optionally substituted aralkyl, —Si(R⁷)₃, —C(O)R⁴, —CO₂R⁴, or—C(O)(NR⁸)R⁴;

R² represents independently for each occurrence H, alkyl, or halogen;

R³, R⁴, and R⁷ each represent independently for each occurrence alkyl,aryl, or aralkyl;

R⁵ is —Si(R⁷)₃, —C(O)R⁴, —CO₂R⁴, or —C(O)(NR⁸)R⁴;

R⁸ represents independently for each occurrence H, alkyl, aryl, oraralkyl;

R⁹ represents independently for each occurrence H or alkyl; and

the stereochemical configuration at any stereocenter of a compoundrepresented by I is R, S, or a mixture of these configurations.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R¹ is optionally substituted aralkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R¹ is optionally substituted trityl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R¹ is optionally substituteddimethoxytrityl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R¹ is

In certain embodiments, the present invention relates to theaforementioned compound, wherein R² is H.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R³ is alkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R³ is methyl, ethyl, propyl, isopropyl,butyl, sec-butyl, isobutyl, tert-butyl, or pentyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R³ is methyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R⁴ is alkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R⁴ represents independently for eachoccurrence methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl,tert-butyl, or pentyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R⁴ is isopropyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R⁵ is Si(R⁷)₃.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R⁵ is Si(R⁷)₃, and R⁷ is alkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R⁵ is Si(CH₃)₂-tert-butyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R⁶ is

In certain embodiments, the present invention relates to theaforementioned compound, wherein R⁶ is

In certain embodiments, the present invention relates to theaforementioned compound, wherein R⁵ is Si(R⁷)₃, and R³, R⁴, and R⁷ arealkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R⁵ is Si(R⁷)₃, and R³, R⁴, and R⁷ arealkyl; R¹ is optionally substituted dimethoxytrityl; and R⁶ is

In certain embodiments, the present invention relates to theaforementioned compound, wherein R² is H, R³ is methyl, R⁴ is isopropyl,R⁵ is Si(CH₃)₂-tert-butyl, R¹ is optionally substituted dimethoxytrityl,and R⁶ is

In certain embodiments, the present invention relates to theaforementioned compound, wherein R² is H, R³ is methyl, R⁴ is isopropyl,R⁵ is Si(CH₃)₂-tert-butyl, R¹ is

and R⁶ is

In certain embodiments, the present invention relates to theaforementioned compound, wherein said compound is represented by formulaIa:

In certain embodiments, the present invention relates to theaforementioned compound, wherein R⁵ is Si(R⁷)₃, and R³, R⁴, and R⁷ arealkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R⁵ is Si(R⁷)₃, and R³, R⁴, and R⁷ arealkyl; R¹ is optionally substituted dimethoxytrityl; and R⁶ is

In certain embodiments, the present invention relates to theaforementioned compound, wherein R² is H, R³ is methyl, R⁴ is isopropyl,R⁵ is Si(CH₃)₂-tert-butyl, R¹ is optionally substituted dimethoxytrityl,and R⁶ is

In certain embodiments, the present invention relates to theaforementioned compound, wherein R² is H, R³ is methyl, R⁴ is isopropyl,R⁵ is Si(CH₃)₂-tert-butyl, R¹ is

and R⁶ is

Another aspect of the present invention relates to an oligonucleotidebearing at least one non-phosphate linkage. In certain embodiments, thenon-phosphate linkage is a phosphonate. In a preferred embodiment, thephosphonate linkage is an alkyl phosphonate. The phosphonate linkagerenders the oligonucleotide less prone to degradation in vivo. Incertain instances, the oligonucleotide is substituted with a ligand. Incertain instances, the ligand is an aralkyl group. The aralkyl ligandrenders the oligonucleotide compound less prone to degradation bynucleases present in the serum, liver, brain, and eye. In certainembodiments, the compounds of the invention relate to a double-strandedoligonucleotide sequence, wherein the aralkyl ligand is bound to onlyone of the two strands. In certain embodiments, the compounds of theinvention relate to a double-stranded oligonucleotide sequence, whereinat least one aralkyl ligand is bound to both of the strands. In certainembodiments, the backbone of the oligonucleotide has been modified toimprove the therapeutic or diagnostic properties of the oligonucleotide.In certain embodiments, at least one of the bases or at least one of thesugars of the oligonucleotide has been modified to improve thetherapeutic or diagnostic properties of the oligonucleotide. The twostrands of the oligonucleotide are complementary or partiallycomplementary. Either strand or both strands may comprise a chimericoligonucleotide. In certain instances, the oligonucleotide is an siRNAagent.

The siRNA agent includes a region of sufficient homology to the targetgene, and is of sufficient length in terms of nucleotides, such that thesiRNA agent, or a fragment thereof, can mediate down-regulation of thetarget gene. It will be understood that the term “ribonucleotide” or“nucleotide” can, in the case of a modified RNA or nucleotide surrogate,also refer to a modified nucleotide, or surrogate replacement moiety atone or more positions. Thus, the siRNA agent is or includes a regionwhich is at least partially complementary to the target RNA. In certainembodiments, the siRNA agent is fully complementary to the target RNA.It is not necessary that there be perfect complementarity between thesiRNA agent and the target, but the correspondence must be sufficient toenable the siRNA agent, or a cleavage product thereof, to directsequence specific silencing, such as by RNAi cleavage of the target RNA.Complementarity, or degree of homology with the target strand, is mostcritical in the antisense strand. While perfect complementarity,particularly in the antisense strand, is often desired some embodimentscan include one or more but preferably 6, 5, 4, 3, 2, or fewermismatches with respect to the target RNA. The mismatches are mosttolerated in the terminal regions, and if present are preferably in aterminal region or regions, e.g., within 6, 5, 4, or 3 nucleotides ofthe 5′ and/or 3′ terminus. The sense strand need only be sufficientlycomplementary with the antisense strand to maintain the overalldouble-strand character of the molecule.

In addition, a siRNA agent will often be modified or include nucleosidesurrogates. Single stranded regions of an siRNA agent will often bemodified or include nucleoside surrogates, e.g., the unpaired region orregions of a hairpin structure, e.g., a region which links twocomplementary regions, can have modifications or nucleoside surrogates.Modification to stabilize one or more 3′- or 5′-terminus of an siRNAagent, e.g., against exonucleases, or to favor the antisense siRNA agentto enter into RISC are also favored. Modifications can include C3 (orC6, C7, C12) amino linkers, thiol linkers, carboxyl linkers,non-nucleotidic spacers (C3, C6, C9, C12, abasic, triethylene glycol,hexaethylene glycol), special biotin or fluorescein reagents that comeas phosphoramidites and that have another DMT-protected hydroxyl group,allowing multiple couplings during RNA synthesis.

siRNA agents include: molecules that are long enough to trigger theinterferon response (which can be cleaved by Dicer (Bernstein et al.2001. Nature, 409:363-366) and enter a RISC (RNAi-induced silencingcomplex)); and, molecules which are sufficiently short that they do nottrigger the interferon response (which molecules can also be cleaved byDicer and/or enter a RISC), e.g., molecules which are of a size whichallows entry into a RISC, e.g., molecules which resemble Dicer-cleavageproducts. Molecules that are short enough that they do not trigger aninterferon response are termed siRNA agents or shorter iRNA agentsherein. “siRNA agent or shorter siRNA agent” as used refers to an siRNAagent that is sufficiently short that it does not induce a deleteriousinterferon response in a human cell, e.g., it has a duplexed region ofless than 60 but preferably less than 50, 40, or 30 nucleotide pairs.The siRNA agent, or a cleavage product thereof, can down regulate atarget gene, e.g., by inducing RNAi with respect to a target RNA,preferably an endogenous or pathogen target RNA.

Each strand of a siRNA agent can be equal to or less than 30, 25, 24,23, 22, 21, or 20 nucleotides in length. The strand is preferably atleast 19 nucleotides in length. For example, each strand can be between21 and 25 nucleotides in length. Preferred siRNA agents have a duplexregion of 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs, andone or more overhangs, preferably one or two 3′ overhangs, of 2-3nucleotides.

In addition to homology to target RNA and the ability to down regulate atarget gene, an siRNA agent will preferably have one or more of thefollowing properties:

(1) it will, despite modifications, even to a very large number, or allof the nucleosides, have an antisense strand that can present bases (ormodified bases) in the proper three dimensional framework so as to beable to form correct base pairing and form a duplex structure with ahomologous target RNA which is sufficient to allow down regulation ofthe target, e.g., by cleavage of the target RNA;

(2) it will, despite modifications, even to a very large number, or allof the nucleosides, still have “RNA-like” properties, i.e., it willpossess the overall structural, chemical and physical properties of anRNA molecule, even though not exclusively, or even partly, ofribonucleotide-based content. For example, an siRNA agent can contain,e.g., a sense and/or an antisense strand in which all of the nucleotidesugars contain e.g., 2′ fluoro in place of 2′ hydroxyl. Thisdeoxyribonucleotide-containing agent can still be expected to exhibitRNA-like properties. While not wishing to be bound by theory, theelectronegative fluorine prefers an axial orientation when attached tothe C2′ position of ribose. This spatial preference of fluorine can, inturn, force the sugars to adopt a C_(3′)-endo pucker. This is the samepuckering mode as observed in RNA molecules and gives rise to theRNA-characteristic A-family-type helix. Further, since fluorine is agood hydrogen bond acceptor, it can participate in the same hydrogenbonding interactions with water molecules that are known to stabilizeRNA structures. Generally, it is preferred that a modified moiety at the2′ sugar position will be able to enter into H-bonding which is morecharacteristic of the OH moiety of a ribonucleotide than the H moiety ofa deoxyribonucleotide. A preferred siRNA agent will: exhibit aC_(3′)-endo pucker in all, or at least 50, 75, 80, 85, 90, or 95% of itssugars; exhibit a C₃′-endo pucker in a sufficient amount of its sugarsthat it can give rise to a the RNA-characteristic A-family-type helix;will have no more than 20, 10, 5, 4, 3, 2, or 1 sugar which is not aC₃′-endo pucker structure.

A “single strand iRNA agent” as used herein, is an iRNA agent which ismade up of a single molecule. It may include a duplexed region, formedby intra-strand pairing, e.g., it may be, or include, a hairpin orpan-handle structure. Single strand iRNA agents are preferably antisensewith regard to the target molecule. A single strand iRNA agent should besufficiently long that it can enter the RISC and participate in RISCmediated cleavage of a target mRNA. A single strand iRNA agent is atleast 14, and more preferably at least 15, 20, 25, 29, 35, 40, or 50nucleotides in length. It is preferably less than 200, 100, or 60nucleotides in length.

Hairpin iRNA agents will have a duplex region equal to or at least 17,18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplex regionwill preferably be equal to or less than 200, 100, or 50, in length.Preferred ranges for the duplex region are 15-30, 17 to 23, 19 to 23,and 19 to 21 nucleotides pairs in length. The hairpin will preferablyhave a single strand overhang or terminal unpaired region, preferablythe 3′, and preferably of the antisense side of the hairpin. Preferredoverhangs are 2-3 nucleotides in length.

Chimeric oligonucleotides, or “chimeras,” are oligonucleotides whichcontain two or more chemically distinct regions, each made up of atleast one monomer unit, i.e., a nucleotide in the case of anoligonucleotide compound. These oligonucleotides typically contain atleast one region wherein the oligonucleotide is modified so as to conferupon the oligonucleotide increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget nucleic acid. Consequently, comparable results can often beobtained with shorter oligonucleotides when chimeric oligonucleotidesare used. Chimeric oligonucleotides of the invention may be formed ascomposite structures of two or more oligonucleotides, modifiedoligonucleotides, oligonucleosides and/or oligonucleotide mimetics asdescribed above. Such oligonucleotides have also been referred to in theart as hybrids or gapmers. Representative United States patents thatteach the preparation of such hybrid structures include, but are notlimited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775;5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355;5,652,356; 5,700,922; and 5,955,589, each of which is hereinincorporated by reference. In certain embodiments, the chimericoligonucleotide is RNA-DNA, DNA-RNA, RNA-DNA-RNA, DNA-RNA-DNA, orRNA-DNA-RNA-DNA, wherein the oligonucleotide is between 5 and 60nucleotides in length.

For the purposes of illustration, a nucleotide bearing an ligand can bedivided into four regions: ligand, tether, linker, and oligonucleotide.The ligand is bound to the oligonucleotide via a tether and linker. Thepurpose of the tether is to covalently attach the ligand, or astructural derivative to the linker. The structure of the tether isdictated by the functional group used to bind the ligand. On the otherhand, the linker serves to bond covalently the oligonucleotide to thetether. In a preferred embodiment, the linker is amenable to solid-phasesynthesis techniques. A more detailed discussion of each of the variableregions presented below.

Ligand

In the present invention, the ligand is an aromatic group, aralkylgroup, or the radical of a steroid, bile acid, lipid, folic acid,pyridoxal, B12, riboflavin, biotin, polycyclic compound, crown ether,intercalator, cleaver molecule, protein-binding agent, carbohydrate, oran optionally substituted saturated 5-membered ring. In certaininstances, the ligand is an aralkyl group, e.g., a 2-arylpropanoylmoiety. The structural features of the ligand are selected so that theligand will bind to at least one protein in vivo. In certainembodiments, the structural features of the ligand are selected so thatligand binds to serum, vascular, or cellular proteins. In certainembodiments, the structural features of the ligand promote binding toalbumin, an immunoglobulin, a lipoprotein, α-2-macroglubulin, orα-1-glycoprotein.

A large number of steroids are known in the art and are amenable to thepresent invention. Representative examples of steriods includecholesterol, 5β-cholanic acid, progesterone, aldosterone,dehydroaldosterone, isoandrosterone, esterone, estradiol, ergosterol,dehydroergosterol, lanosterol, 4-cholesten-3-one, guggulsterone,testosterone, nortestosterone, formestane, hydroxyecdysone, ketoestriol,corticosterone, dienestrol, dihydroxypregnanone, pregnanone, copornmon,equilenin, equilin, estriol, ethinylestradiol, mestranol, moxestrol,mytatrienediol, quinestradiol, quinestrol, helvolic acid, protostadiene,fusidic acid, cycloartenol, tricallol, cucurbitanin cedrelone, euphol,dammerenediol, parkeol, dexametasone, methylprednisolone, prednisolone,hydrocortisone, parametasone, betametasone, cortisone, fluocinonide,fluorometholone, halcinonide, and budesonide, or any one of them furthersubstituted with one or more of hydroxyl, halogen, amino, alkylamino,alkyl, carboxylic acid, ester, amide, carbonyl, alkoxyl, or cyano.

A large number of bile acids are known in the art and are amenable tothe present invention. Bile acids occur in conjugation with glycine ortaurine in bile of most vertebrates and some of them find use inmedicine. Thus, some bile acids—due to their inherent pharmacologicalproperties—are used as cholerectics (see, for example, James E. F.Reynolds (editor) Martindale The Extra Pharmacopoeia, 30^(th) Edition,The Pharmaceutical Press, London (1993), page 1341). Representativeexamples of bile acids include cholic acid, deoxycholic acid,taurocholic acid, glycocholic acid, glycodeoxycholic acid,taurodeoxycholic acid, ursodeoxycholic acid, and chenodeoxycholic acid.Additional bile acids amenable to the present invention include thosedescribed in U.S. Pat. Nos. 5,641,767; 5,656,277; 5,610,151; 5,428,182;and 3,910,888.

A large number of lipids are known in the art and are amenable to thepresent invention. Representative examples of lipids include lauricacid, myristic acid, palmitic acid, stearic acid, arachidic acid,palmitoleic acid, oleic acid, linoleic acid, linolenic acid, arachidonicacid, triacylglycerols, phosphoacylglycerols, sphingolipids,monoterpenes, sesquiterpenes, diterpenes, sesterterpenes, triterpenes,and tetraterpenes.

A large number of aromatic compounds are known in the art and areamenable to the present invention. Representative examples of aromaticcompounds include optionally substituted phenyl, naphthyl, anthracenyl,phenanthrenyl, pyrenyl, pyridinyl, quinolinyl, acridinyl,phenathridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinoxalinyl,quinazolinyl, 1,7-phenanthrolinyl, indolyl, thianaphthenyl,benzoxazolyl, benzofuranyl, 1,2-benzisoxazolyl, benzimidazolyl,pyrrolyl, thiophenyl, isoxazolyl, pyrazolyl, thiazolyl, imidazolyl,tetrazolyl, and furanyl.

A large number of carbohydrates are known in the art and are amenable tothe present invention. Representative examples of carbohydrates includeerythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose,glucose, mannose, gulose, idose, galactose, and talose; or adisaccharide or trisaccharide formed via a 1,4 glycoside linkage betweenany of them. In certain instances, the carbohydrate is a hexose orpentose.

A large number of polycyclic compounds are known in the art and areamenable to the present invention. Representative classes of polycycliccompounds include bicyclic compounds wherein, the first and second ringare independently a 3, 4, 5, or 6-member saturated or unsaturated carbonring containing 0, 1, 2, or 3 hetereoatoms selected from the groupconsisting of O, N, or S. In certain instances, the first ring is anaromatic ring. In certain instances, the second ring is an aromaticring. In certain instances, both rings are saturated. In certaininstances, the first ring contains no heteroatoms. In certain instances,the second ring contains to heteroatoms. In certain instances, the firstring contains a nitrogen atom. In certain instances, the second ringcontains a nitrogen atom. In certain instances, the polycyclic compoundis a tricyclic compound, wherein the first, second, and third ring areindependently a 3, 4, 5, or 6-member saturated or unsaturated carbonring containing 0, 1, 2, or 3 hetereoatoms selected from the groupconsisting of O, N, or S. In certain instances, the first ring is anaromatic ring. In certain instances, the second ring is an aromaticring. In certain instances, the third ring is an aromatic ring. Incertain instances, all three rings are saturated. In certain instances,the first ring contains no heteroatoms. In certain instances, the secondring contains to heteroatoms. In certain instances, the third ringcontains to heteroatoms. In certain instances, the first ring contains anitrogen atom. In certain instances, the second ring contains a nitrogenatom. In certain instances, the third ring contains a nitrogen atom. Incertain instances, the polycyclic compound is a bridged polycycliccompound. In certain instances, the polycyclic compound is abicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[3.2.1]octane,bicyclo[3.2.2]nonane, or bicyclo[3.3.1]nonane.

A large number of crown ethers are known in the art and are amenable tothe present invention. Crown ethers are macrocyclic, polyether, neutralcompounds containing 4-20 oxygen atoms each separated from the next bytwo or more carbon atoms. Macrocyclic polyethers have been found to formstable complexes with salts of alkali metals and other metals andammonium salts; “Macrocyclic polyethers and their complexes”, C. J.Pederson et al, Angew. Chem. Intern. Ed., Vol. 11, page 16, (1972) andU.S. Pat. Nos. 3,562,295 and 3,687,978. Since the stereo models ofmacrocyclic polyethers give a crown-like appearance, they are commonlydesignated as N-crown-M polyethers, wherein N is the total number ofatoms in the polyether ring and M is the number of oxygen atoms in thepolyether ring. Crown polyethers ranging in size from cyclic tetramersof ethylene oxide ([12]-crown-4) and propylene oxide ([16]-crown-4) to60-membered polyether rings (dibenzo [60]-crown-20) have been reported.Preferred crown ethers include 12-crown-4, 15-crown-5, and 18-crown-6.

A large number of oligonucleotide intercalators are known in the art andare amenable to the present invention. One class of intercalators areDNA intercalators which bind noncovalently to duplex DNA and arecharacterized by a flat molecule which inserts between base pairs of thedouble helix of DNA. Representative examples of intercalators includep-carboxy methidium, p-carboxy ethidium, acridine and ellipticine.

A large number of oligonucleotide cleaver molecules are known in the artand are amenable to the present invention. A cleaver molecule is acompound that can sever an oligonucleotide strand. Bleomycin, aglycopeptide antibiotic, is known to bind to and cleave DNA in areaction that depends on the presence of ferrous ion and molecularoxygen, “Bleomycin: Chemical, Biochemical and Biological Aspects”;Hecht, S. M., Ed.; Springer Verlag: New York, 1979; Sausville, E. A.;Peisach, J.; Horwitz, S. B. “Biochemistry” 1978, 17, 2740. Burger, R.M.; Peisach, J; Horwitz, S. B. “Life Sciences” 1981, 28, 715; and Lown,J. W.; Sim, S. F. “Biochem. Biophys. Res. Comm.” 1977, 77, 1150. Theantitumor agent streptonigrin is also capable of causing single strandbreaks in DNA using oxygen and cuprous ion, Cone, R; Hasan, S. K.; Lown,J. W.; Morgan, A. R. “Can. J. Biochem.” 1976, 54, 219. Recently, the1-10 phenanthroline-cuprous complex has been shown to cleave DNA in thepresence of oxygen, Sigman, D. S.; Graham, D. R.; D'Aurora, V.; Stern,A. M. “J. Biol. Chem.” 1979, 254, 12269; Graham, D. R.; Marshall, L. E.;Reich, K. A.; Sigman, D. S. “J. Amer. Chem. Soc.” 1980, 102, 5419;Marshall, L. E.; Graham, D. R.; Reich, K. A.; Sigman, D. S.“Biochemistry” 1981, 20, 244; and Que, B. G.; Downey, K. M.; So., A. G.“Biochemistry” 1980, 19, 5987. In addition, methidium, ethidium, andcisplatin are known to cleave oligonucleotide sequences.

A large number of saturated 5-membered rings are known in the art andare amenable to the present invention. Preferred saturated 5-memberedrings are optionally substituted cyclopentane, pyrrolidine,tetrahydrofuran, tetrahydrothiophene, and 1,1-difluorocyclopentane.

In certain instances, the oligonucleotides of the invention contain atleast one nucleoside that is bound to a ligand. In certain instances,there are 3, 4, 5, 10, or 15 nucleotides that are individuallycovalently bonded to separate ligands. In certain instances, the ligandis bonded to the 5′-position or the 3′-position of the terminalnucleoside. In certain instances, an aralkyl ligand is bonded to boththe 5′-position and the 3′-position of the terminal nucleoside. Incertain instances, a ligand is bonded to both the 3′-position of thenucleoside located at the 3′-terminus of the oligonucleotide. In certaininstances, the linker forms a covalent linkage between two nucleosidesand the linker is also bonded to the ligand via a tether. In certaininstances, a hairpin structure is formed when the linker forms acovalent linkage between two nucleosides and the linker is also bondedto the ligand via a tether. In certain instances, more than one ligandis bonded to the tether. FIGS. 1 and 10 illustrate several ways in whichthe ligand is attached to the oligonucleotide.

In certain embodiments, the ligand is naproxen or a structuralderivative of naproxen. Procedures for the synthesis of naproxen can befound in U.S. Pat. No. 3,904,682 and U.S. Pat. No. 4,009,197. Naproxenhas the chemical name (S)-6-Methoxy-α-methyl-2-naphthaleneacetic acidand the structure is shown below.

In certain embodiments, the ligand is ibuprofen or a structuralderivative of ibuprofen. Procedures for the synthesis of ibuprofen canbe found in U.S. Pat. No. 3,228,831. The structure of ibuprofen is shownbelow.

Various additional ligands are presented in FIGS. 3-9.

Oligonucleotide & Linker

The nucleosides are linked by phosphorus-containing ornon-phosphorus-containing covalent internucleoside linkages. For thepurposes of identification, such conjugated nucleosides can becharacterized as ligand-bearing nucleosides or ligand-nucleosideconjugates. The linked nucleosides having an aralkyl ligand conjugatedto a nucleoside within their sequence will demonstrate enhanced siRNAactivity when compared to like siRNA compounds that are not conjugated.

The ligand-conjugated oligonucleotides of the present invention alsoinclude conjugates of oligonucleotides and linked nucleosides whereinthe ligand is attached directly to the nucleoside or nucleotide withoutthe intermediacy of a linker group. The ligand may preferably beattached, via linking groups, at a carboxyl, amino or oxo group of theligand. Typical linking groups may be ester, amide or carbamate groups.

The oligonucleotides of the present invention have been chemicallymodified. A variety of specific oligonucleotide chemical modificationsare described below. Importantly, it is not necessary for all positionsin a given compound to be uniformly modified. Conversely, more than onemodifications may be incorporated in a single siRNA compound or even ina single nucleotide thereof.

In certain instances, both the sugar and the internucleoside linkage,i.e., the backbone, of the nucleoside units are replaced with novelgroups. The nucleobase units are maintained for hybridization with anappropriate nucleic acid target compound. One such oligonucleotide, anoligonucleotide mimetic, that has been shown to have excellenthybridization properties, is referred to as a peptide nucleic acid(PNA). In PNA compounds, the sugar-backbone of an oligonucleotide isreplaced with an amide-containing backbone, in particular anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to atoms of the amide portion of the backbone.Representative United States patents that teach the preparation of PNAcompounds include, but are not limited to, U.S. Pat. Nos. 5,539,082;5,714,331; and 5,719,262, each of which is herein incorporated byreference. Further teaching of PNA compounds can be found in Nielsen etal., Science, 1991, 254, 1497.

The oligonucleotides of the present invention may additionally oralternatively comprise nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C), anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808,those disclosed in the Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligonucleotides of the invention. These include 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-Methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Id., pages 276-278) and are presentlypreferred base substitutions, even more particularly when combined with2′-methoxyethyl sugar modifications.

Representative United States patents relating to the preparation ofcertain of the above-noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,681,941; and 5,808,027; all of which are herebyincorporated by reference.

In certain embodiments, the oligonucleotides of the present inventionmay additionally or alternatively comprise one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; O-, S-, or N-alkyl, O-, S-, or N-alkenyl, or O,S- or N-alkynyl, wherein the alkyl, alkenyl and alkynyl may besubstituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl andalkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃,SOCH₃, SO₂ CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. apreferred modification includes 2′-methoxyethoxy [2′-O—CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE] (Martin et al., Helv. Chim.Acta, 1995, 78, 486), i.e., an alkoxyalkoxy group. a further preferredmodification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂group, also known as 2′-DMAOE, as described in U.S. Pat. No. 6,127,533,filed on Jan. 30, 1998, the contents of which are incorporated byreference.

Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides.

As used herein, the term “sugar substituent group” or “2′-substituentgroup” includes groups attached to the 2′-position of the ribofuranosylmoiety with or without an oxygen atom. Sugar substituent groups include,but are not limited to, fluoro, O-alkyl, O-alkylamino, O-alkylalkoxy,protected O-alkylamino, O-alkylaminoalkyl, O-alkyl imidazole andpolyethers of the formula (O-alkyl)_(m), wherein m is 1 to about 10.Preferred among these polyethers are linear and cyclic polyethyleneglycols (PEGs), and (PEG)-containing groups, such as crown ethers andthose which are disclosed by Ouchi et al. (Drug Design and Discovery1992, 9:93); Ravasio et al. (J. Org. Chem. 1991, 56:4329); and Delgardoet. al. (Critical Reviews in Therapeutic Drug Carrier Systems 1992,9:249), each of which is hereby incorporated by reference in itsentirety. Further sugar modifications are disclosed by Cook (Anti-CancerDrug Design, 1991, 6:585-607). Fluoro, O-alkyl, O-alkylamino, O-alkylimidazole, O-alkylaminoalkyl, and alkyl amino substitution is describedin U.S. Pat. No. 6,166,197, entitled “Oligomeric Compounds havingPyrimidine Nucleotide(s) with 2′ and 5′ Substitutions,” herebyincorporated by reference in its entirety.

Additional sugar substituent groups amenable to the present inventioninclude 2′-SR and 2′-NR₂ groups, wherein each R is, independently,hydrogen, a protecting group or substituted or unsubstituted alkyl,alkenyl, or alkynyl. 2′-SR Nucleosides are disclosed in U.S. Pat. No.5,670,633, hereby incorporated by reference in its entirety. Theincorporation of 2′-SR monomer synthons is disclosed by Hamm et al. (J.Org. Chem., 1997, 62:3415-3420). 2′-NR nucleosides are disclosed byGoettingen, M., J. Org. Chem., 1996, 61, 6273-6281; and Polushin et al.,Tetrahedron Lett., 1996, 37, 3227-3230. Further representative2′-substituent groups amenable to the present invention include thosehaving one of formula I or II:

wherein,

E is C₁-C₁₀ alkyl, N(Q₃)(Q₄) or N═C (Q₃)(Q₄); each Q₃ and Q₄ is,independently, H, C₁-C₁₀ alkyl, dialkylaminoalkyl, a nitrogen protectinggroup, a tethered or untethered conjugate group, a linker to a solidsupport; or Q₃ and Q₄, together, form a nitrogen protecting group or aring structure optionally including at least one additional heteroatomselected from N and O;

q₁ is an integer from 1 to 10;

q₂ is an integer from 1 to 10;

q₃ is 0 or 1;

q₄ is 0, 1 or 2;

each Z₁, Z₂ and Z₃ is, independently, C₄-C₇ cycloalkyl, C₅-C₁₄ aryl orC₃-C₁₅ heterocyclyl, wherein the heteroatom in said heterocyclyl groupis selected from oxygen, nitrogen and sulfur;

Z₄ is OM₁, SM₁, or N(M₁)₂; each M₁ is, independently, H, C₁-C₈ alkyl,C₁-C₈ haloalkyl, C(═NH)N(H)M₂, C(═O)N(H)M₂ or OC(═O)N(H)M₂; M₂ is H orC₁-C₈ alkyl; and

Z₅ is C₁-C₁₀ alkyl, C₁-C₁₀ haloalkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl,C₆-C₁₄ aryl, N(Q₃)(Q₄), OQ₃, halo, SQ₃ or CN.

Representative 2′-O-sugar substituent groups of formula I are disclosedin U.S. Pat. No. 6,172,209, entitled “Capped 2′-OxyethoxyOligonucleotides,” hereby incorporated by reference in its entirety.Representative cyclic 2′-O-sugar substituent groups of formula II aredisclosed in U.S. Pat. No. 6,271,358, entitled “RNA Targeted 2′-ModifiedOligonucleotides that are Conformationally Preorganized,” herebyincorporated by reference in its entirety.

Sugars having O-substitutions on the ribosyl ring are also amenable tothe present invention. Representative substitutions for ring O include,but are not limited to, S, CH₂, CHF, and CF₂. See, e.g., Secrist et al.,Abstract 21, Program & Abstracts, Tenth International Roundtable,Nucleosides, Nucleotides and their Biological Applications, Park City,Utah, Sep. 16-20, 1992.

Oligonucleotides may also have sugar mimetics, such as cyclobutylmoieties, in place of the pentofuranosyl sugar. Representative UnitedStates patents relating to the preparation of such modified sugarsinclude, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800;5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785;5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300;5,627,0531 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,700,920; and5,859,221, all of which are hereby incorporated by reference.

Additional modifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide. For example, one additional modification of theligand-conjugated oligonucleotides of the present invention involveschemically linking to the oligonucleotide one or more additionalnon-ligand moieties or conjugates which enhance the activity, cellulardistribution or cellular uptake of the oligonucleotide. Such moietiesinclude but are not limited to lipid moieties, such as a cholesterolmoiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553),cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053),a thioether, e.g., hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y.Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let.,1993, 3, 2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.,1992, 20, 533), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., EMBO J, 1991, 10, 111; Kabanov etal., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75,49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990,18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14, 969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277, 923).

Representative United States patents relating to the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928; and 5,688,941, each of whichis herein incorporated by reference.

The present invention also includes compositions employingoligonucleotides that are substantially chirally pure with regard toparticular positions within the oligonucleotides. Examples ofsubstantially chirally pure oligonucleotides include, but are notlimited to, those having phosphorothioate linkages that are at least 75%Sp or Rp (Cook et al., U.S. Pat. No. 5,587,361) and those havingsubstantially chirally pure (Sp or Rp) alkylphosphonate, phosphoramidateor phosphotriester linkages (Cook, U.S. Pat. Nos. 5,212,295 and5,521,302).

The present invention further encompasses oligonucleotides employingribozymes. Synthetic RNA molecules and derivatives thereof that catalyzehighly specific endoribonuclease activities are known as ribozymes.(See, generally, U.S. Pat. No. 5,543,508 to Haseloff et al., and U.S.Pat. No. 5,545,729 to Goodchild et al.) The cleavage reactions arecatalyzed by the RNA molecules themselves. In naturally occurring RNAmolecules, the sites of self-catalyzed cleavage are located withinhighly conserved regions of RNA secondary structure (Buzayan et al.,Proc. Natl. Acad. Sci. U.S.A., 1986, 83, 8859; Forster et al., Cell,1987, 50, 9). Naturally occurring autocatalytic RNA molecules have beenmodified to generate ribozymes which can be targeted to a particularcellular or pathogenic RNA molecule with a high degree of specificity.Thus, ribozymes serve the same general purpose as antisenseoligonucleotides (i.e., modulation of expression of a specific gene)and, like oligonucleotides, are nucleic acids possessing significantportions of single-strandedness. That is, ribozymes have substantialchemical and functional identity with oligonucleotides and are thusconsidered to be equivalents for purposes of the present invention.

In certain instances, the oligonucleotide may be modified by anon-ligand group. A number of non-ligand molecules have been conjugatedto oligonucleotides in order to enhance the activity, cellulardistribution or cellular uptake of the oligonucleotide, and proceduresfor performing such conjugations are available in the scientificliterature. Such non-ligand moieties have included lipid moieties, suchas cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989,86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994,4:1053), a thioether, e.g., hexyl-5-tritylthiol (Manoharan et al., Ann.N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem.Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. AcidsRes., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov etal., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993,75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl.Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923). Representative United States patents thatteach the preparation of such oligonucleotide conjugates have beenlisted above. Typical conjugation protocols involve the synthesis ofoligonucleotides bearing an aminolinker at one or more positions of thesequence. The amino group is then reacted with the molecule beingconjugated using appropriate coupling or activating reagents. Theconjugation reaction may be performed either with the oligonucleotidestill bound to the solid support or following cleavage of theoligonucleotide in solution phase. Purification of the oligonucleotideconjugate by HPLC typically affords the pure conjugate.

Alternatively, the molecule being conjugated may be converted into abuilding block, such as a phosphoramidite, via an alcohol group presentin the molecule or by attachment of a linker bearing an alcohol groupthat may be phosphitylated.

Importantly, each of these approaches may be used for the synthesis ofligand conjugated oligonucleotides. Aminolinked oligonucleotides may becoupled directly with ligand via the use of coupling reagents orfollowing activation of the ligand as an NHS or pentfluorophenolateester. Ligand phosphoramidites may be synthesized via the attachment ofan aminohexanol linker to one of the carboxyl groups followed byphosphitylation of the terminal alcohol functionality. Other linkers,such as cysteamine, may also be utilized for conjugation to achloroacetyl linker present on a synthesized oligonucleotide.

Tether

In a preferred embodiment of the invention, the ligand is attached to anoligonucleotide via a tether and linking group, to form aligand-conjugated oligonucleotide. Preferred tethers of the inventioninclude, but are not limited to, 6-aminoalkoxy linkers,6-aminoalkylamino linkers, cysteamine, heterobifunctional linkers,homobifunctional linkers, and a universal tether (derived from3-dimethoxytrityloxy-2-aminopropanol). A particularly preferred tetherfor the synthesis of ligand conjugated oligonucleotides of the inventionis a 6-aminohexyloxy group. A variety of heterobifunctional andhomobifunctional tethers are available from Pierce Co. (Rockford, Ill.).Such heterobifunctional and homobifunctional tethers are particularlyuseful in conjunction with the 6-aminoalkoxy and 6-aminoalkylaminomoieties to form extended tethers useful for linking ligands to anucleoside. Further useful tethers that are commercially available are5′-Amino-Modifier C6 and 3′-Amino-Modifier reagents, both available fromGlen Research Corporation (Sterling, Va.). 5′-Amino-Modifier C6 is alsoavailable from ABI (Applied Biosystems Inc., Foster City, Calif.) asAminolink-2, while the 3′-Amino-Modifier is also available from ClontechLaboratories Inc. (Palo Alto, Calif.). In addition, a nucleotide analogbearing a tether pre-attached to the nucleoside is commerciallyavailable from Glen Research Corporation under the tradename“Amino-Modifier-dT.” This nucleoside-tether reagent, a uridinederivative having an [N(7-trifluoroacetylamino-heptyl)3-acrylamido]substituent group at the 5 position of the pyrimidine ring, issynthesized as per the procedure of Jablonski et al. (Nucleic AcidResearch, 1986, 14:6115).

In certain instances, conjugation of ligand molecules is achieved byconjugation of the ligand to an amino tether on the nucleoside. This canbe effected in several ways. For example, a ligand-nucleoside conjugateof the invention can be prepared by conjugation of the ligand moleculeto the nucleoside using EDC/sulfo-NHS (i.e.,1-ethyl-3(3-dimethylaminopropylcarbodiimide/N-hydroxysulfosuccinimide)to conjugate the carboxylate function of the ligand with the aminofunction of the linking group on the nucleoside.

The ligand-conjugated oligonucleotides of the present invention may beprepared by conjugation of the ligand (e.g., naproxen) molecule to thenucleoside sequence via a heterobifunctional tether such asm-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (MBS) or succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), to link anucleophilic position on the ligand molecule to the amino function ofthe tether group on nucleoside sequence. By this mechanism, anoligonucleoside-maleimide conjugate is formed by reaction of the aminogroup of the tether on the linked nucleosides with the MBS or SMCCmaleimide linker. The conjugate is then reacted with the ligand.

Alternatively, a ligand conjugated-oligonucleotide can be prepared byconjugation of the ligand molecule to the oligonucleotide or nucleosidevia a homobifunctional tether such as disuccinimidyl suberate (DSS), tolink an amino function on the ligand to the amino group of a tether onthe oligonucleotide sequence. By this mechanism, anoligonucleoside-succinimidyl conjugate is formed by reaction of theamino group of the tether on the nucleoside sequence with adisuccinimidyl suberate tether. The disuccinimidyl suberate tethercouples with the amine tether on the nucleoside to extend the size ofthe tether. The extended tether is then reacted with an amino group ofthe ligand molecule.

Certain compounds of the invention are described below in greaterdetail. Importantly, the embodiments described below are included merelyfor purposes of illustration of certain aspects and embodiments of thepresent invention, and are not intended to limit the invention.

One aspect of the present invention relates to a double-strandedoligonucleotide comprising a first strand and a second strand, whereinsaid first strand and said second strand are represented independentlyby formula II:

wherein

X¹ is H, —P(O)(OM)₂, —P(O)(OM)-O—P(O)(OM)₂, —P(O)(Oalkyl)₂,—P(O)(Oalkyl)-O—P(O)(Oalkyl)₂, or -A⁶-[A⁷-(A⁵)_(w)]_(y);

M represents independently for each occurrence an alkali metal or atransition metal with an overall charge of +1;

R¹ and R⁵ represent independently for each occurrence H, alkyl, orhalogen;

R² and R³ represent independently for each occurrence H, OH, F, —Oalkyl,—Oallyl, —O(C(R¹⁹)₂)_(k)OR¹⁹, —O(C(R¹⁹)₂)_(k)SR¹⁹,—O(C(R¹⁹)₂)_(k)N(R¹⁹)₂, —O(C(R¹⁹)₂)_(k)C(O)N(R¹⁹)₂, —N(R¹⁹)₂,—S(C₁-C₆)alkyl, —O(C(R¹⁹)₂)_(k)O(C₁-C₆)alkyl,—O(C(R¹⁹)₂)_(k)S(C₁-C₆)alkyl,—O(C(R¹⁹)₂)_(k)O(C(R¹⁹)₂)_(k)N((C₁-C₆)alkyl)₂,—O(C(R¹⁹)₂)_(k)ON((C₁-C₆)alkyl)₂, or —O-A⁶-[A⁷-(A⁵)_(w)]_(y);

R⁴ represents independently for each occurrence H, OH, F, —Oalkyl,—Oallyl, —O(C(R¹⁹)₂)_(k)OR¹⁹, —O(C(R¹⁹)₂)_(k)SR¹⁹,—O(C(R¹⁹)₂)_(k)N(R¹⁹)₂, —O(C(R¹⁹)₂)_(k)C(O)N(R¹⁹)₂, —N(R¹⁹)₂,—S(C₁-C₆)alkyl, —O(C(R¹⁹)₂)_(k)O(C₁-C₆)alkyl,—O(C(R¹⁹)₂)_(k)S(C₁-C₆)alkyl,—O(C(R¹⁹)₂)_(k)O(C(R¹⁹)₂)_(k)N((C₁-C₆)alkyl)₂, or—O(C(R¹⁹)₂)_(k)ON((C₁-C₆)alkyl)₂;

R⁶, R⁷, and R⁹ represent independently for each occurrence H, alkyl,aryl, or aralkyl;

R⁸ represents independently for each occurrence alkyl, aryl, or aralkyl;

k represents independently for each occurrence 1, 2, 3, or 4;

n¹ is 1, 2, or 3;

n² is an integer in the range of about 15-28, inclusive;

w represents independently for each occurrence 1, 2, or 3 in accord withthe rules of valence;

x represents independently for each occurrence 0, 1, 2, or 3;

y represents independently for each occurrence 1, 2, 3, 4, or 5 inaccord with the rules of valence;

A¹ represents independently for each occurrence:

A² represents independently for each occurrence:

A³ represents independently for each occurrence

A⁴ represents independently for each occurrence a bond, alkyl diradical,heteroalkyl diradical, alkenyl diradical, alkynyl diradical,alkylalkynyl diradical, aminoalkyl diradical, thioether, —C(O)—, —S(O)—,—S(O)₂—, B¹C(R)₂B², B¹C(R)(B²)₂, B¹C(B²)₃, B¹N(R)(B²), B¹N(B²)₂, or hasthe formula:

B¹ is a bond between A³ and A⁴;

B² is a bond between A⁴ and A⁵;

R represents independently for each occurrence hydrogen or alkyl;

m represents independently for each occurrence 1, 2, 3, 4, 5, 6, 7, or8;

m¹ represents independently for each occurrence 0, 1, 2, 3, 4, 5, 6, 7,or 8;

Y represents independently for each occurrence an alkyl diradical,cycloalkyl diradical, heteroalkyl diradical, heterocycloalkyl diradical,alkenyl diradical, alkynyl diradical, aryl diradical, heteroaryldiradical, aralkyl diradical, heteroaralkyl diradical,—X²C(O)X²[C(R⁵)₂]_(v)X²—, —X²C(NR⁶)X²[C(R⁵)₂]_(v)X²—,—X²C(S)X²[C(R⁵)₂]_(v)X²—, —X²C(O)X²[C(R⁵)₂]_(v)X²C(O)X²—,

[C(R⁵)₂]_(t)N(R⁶)O[C(R⁵)₂]_(t)—, —[C(R⁵)₂]_(t)N(R⁶)N(R⁶)O[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)N(R⁷)C(O)[C(R⁵)₂]_(t)—, —[C(R⁵)₂]_(t)N(R)CO₂[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)N(R⁷)C(S)[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)N(R⁷)C(S)O[C(R⁵)₂]_(t)—, —[C(R⁵)₂]_(t)OC(O)S[C(R⁵)₂]_(t)—,—[C(R)₂]_(t)SN(R⁷)CO₂[C(R⁵)₂]_(t)—, —[C(R⁵)₂]_(t)OSi(R⁸)₂O[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)OSO₂N(R⁷)[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)N(R⁷)SO₂N(R⁷)[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)SO₂N(morpholino)-[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)SO₂N(R⁷)[C(R⁵)₂]_(t)—, —[C(R¹)₂]_(t)S[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)OSO₂[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)S[C(R⁵)₂]_(y)O[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)O[C(R⁵)₂]_(y)O[C(R⁵)₂]_(t)—, —[C(R⁵)₂]_(t)O[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)N(R⁷)[C(R⁵)₂]_(t)—, [C(R⁵)₂]_(t)C═NO[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)C(O)C(R⁵═C(R⁵)[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)C(R⁵)═C(R⁵)[C(R⁵)₂]_(t)—, or—[C(R⁵)₂]_(t)X²C(O)X²[C(R⁵)₂]_(t)—;

X² represents independently for each occurrence a bond, O, or N(R⁶);

Z¹ represents independently for each occurrence O, S, or N(R⁸);

Z² represents independently for each occurrence alkyl, aryl, aralkyl,B(R⁹)₃, —OM, —Oalkyl, —Oaryl, —Oaralkyl, —SM, —Salkyl, —Saryl,—Saralkyl, —[C(R⁵)₂]_(m)N(R⁶)₂, —N(R¹⁰)R¹¹, —N(R¹⁹)(C(R¹⁹)₂)_(m)N(R¹⁹)₂,—N(R⁷)C(O)R⁸, H, —OC(O)R⁸, —CO₂R⁸, F, Se, —SeR⁸, —(C(R¹⁹)₂)_(m)OR¹⁹,—(C(R¹⁹)₂)_(m)SR¹⁹, —N(R¹⁹)(C(R¹⁹)₂)_(m)OR¹⁹, —N(R¹⁹)(C(R¹⁹)₂)_(m)SR¹⁹,—N(R¹⁹)(C(R¹⁹)₂)_(m)N(R¹⁹)C(O)alkyl, —(C(R¹⁹)₂)_(m)N(R¹⁹)C(O)alkyl, or-A⁸-[A⁹-(A⁵)_(w)]_(y);

R¹⁰ and R¹¹ are independently H, alkyl, or aryl; or R¹⁰ and R¹¹ takentogether form a 3-, 4-, 5-, 6-, or 7-member ring;

R¹² represents independently for each occurrence H, alkyl or—NHCH₂CH═CH₂;

t represents independently for each occurrence 0, 1, 2, 3, or 4;

v represents independently for each occurrence 0, 1, 2, 3, 4, 5, 6, 7,or 8;

A⁵ represents independently for each occurrence aryl, aralkyl, or theradical of a steroid, bile acid, lipid, folic acid, pyridoxal, B12,riboflavin, biotin, polycyclic compound, crown ether, intercalator,cleaver molecule, protein-binding agent, carbohydrate, or an optionallysubstituted saturated 5-membered ring;

A⁶ represents independently for each occurrence a bond, alkyl diradical,heteroalkyl diradical, alkenyl diradical, aminoalkyl, —C(O)—, —S(O)—,—S(O)₂—, or is represented by formula:

Z³ represents independently for each occurrence O or S;

Z⁴ represents independently for each occurrence —OM, —Oalkyl, —Oaryl,—Oaralkyl, —SM, —Salkyl, —Saryl, —Saralkyl, —N(R¹⁰)R¹¹,—[C(R⁵)₂]_(m)N(R⁶)₂, —N(R¹⁹)(C(R¹⁹)₂)_(m)N(R¹⁹)₂, —(C(R¹⁹)₂)_(m)OR¹⁹,—(C(R¹⁹)₂)_(m)SR¹⁹, —N(R¹⁹)(C(R¹⁹)₂)_(m)OR¹⁹, —N(R¹⁹)(C(R¹⁹)₂)_(m)SR¹⁹,—N(R¹⁹)(C(R¹⁹)₂)_(m)N(R¹⁹)C(O)alkyl, —(C(R¹⁹)₂)_(m)N(R¹⁹)C(O)alkyl,aryl, or alkyl;

R¹³ represent independently for each occurrence H, alkyl, cycloalkyl,heteroalkyl, aryl, aralkyl, acyl, silyl, or B³;

R¹⁴ represents independently for each occurrence alkyl, aryl, aralkyl,acyl, or silyl;

R¹⁵ represents independently for each occurrence hydrogen, alkyl, aryl,aralkyl, acyl, alkylsulfonyl, alkylsulfoxide, arylsulfonyl,arylsulfoxide, or silyl;

R¹⁶ represents independently for each occurrence cycloalkyl,heterocycloalkyl, aryl, or heteroaryl;

B³ is a bond between A⁶ and A⁷;

B⁴ is a bond between A⁶ and O;

n³ represents independently for each occurrence an integer in the rangeof 1-15, inclusive;

n⁴ represents independently for each occurrence 1, 2, 3, 4, or 5 inaccord with the rules of valence;

A⁷ represents independently for each occurrence a bond, alkyl diradical,heteroalkyl diradical, —C(O)—, —S(O)—, —S(O)₂—, B³C(R)₂B⁵, B³C(R)(B⁵)₂,B³C(B⁵)₃, B³N(R)(B⁵), B³N(B⁵)₂, or has the formula:

p represents independently for each occurrence 1, 2, 3, or 4;

B⁵ is a bond between A⁵ and A⁷;

A⁸ is a bond, alkyl diradical, heteroalkyl diradical, alkenyl diradical,aminoalkyl, or is represented by formula:

R¹⁷ represent independently for each occurrence H, alkyl, cycloalkyl,heteroalkyl, aryl, aralkyl, acyl, silyl, or B⁶;

R¹⁸ represents independently for each occurrence H, halogen, alkyl,alkoxyl, —N(R⁶)₂, —CN, —[C(R⁵)₂]_(v)C(R⁵)═C(R⁵)₂;

R¹⁹ represents independently for each occurrence H or alkyl;

B⁶ is a bond between A⁸ and A⁹;

B⁷ is a bond between A⁸ and P;

A⁹ is a bond, alkyl diradical, heteroalkyl diradical, —C(O)—, —S(O)—,—S(O)₂—, B⁶C(R)₂B⁸, B⁶C(R)(B⁸)₂, B⁶C(B⁸)₃, B⁶N(R)(B⁸), B⁶N(B⁸)₂, or hasthe formula:

B⁸ is a bond between A⁵ and A⁹; and

provided that at least one instance of Y is not

In certain embodiments, the present invention relates to theaforementioned compound, wherein A¹ represents independently for eachoccurrence:

In certain embodiments, the present invention relates to theaforementioned compound, wherein Y is

In certain embodiments, the present invention relates to theaforementioned compound, wherein Y is

and Z² represents independently for each occurrence alkyl, aryl,aralkyl, B(R⁹)₃, —OM, —Oalkyl, —Oaryl, or —Oaralkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein Y is

and Z² represents independently for each occurrence alkyl or —OM.

In certain embodiments, the present invention relates to theaforementioned compound, wherein Y is

and Z² represents independently for each occurrence methyl, ethyl,propyl, isopropyl, or —OM.

In certain embodiments, the present invention relates to theaforementioned compound, wherein Y is

and Z² represents independently for each occurrence methyl or —OM.

In certain embodiments, the present invention relates to theaforementioned compound, wherein Y is

and there are at least two instances when Z² is alkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein Y is

and there are at least five instances when Z² is alkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein Y is

and there are at least seven instances when Z² is alkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein Y is

and there are at least ten instances when Z² is alkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein in the linkage between the firstnucleoside and second nucleoside at the terminus of said first strand, Yis

and Z² is alkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein in the linkage between the first andsecond nucleoside at the 3′-terminus of said first strand, Y is

and Z² is alkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein in the linkage between the first andsecond nucleoside at the 3′-terminus of said first strand and saidsecond strand, Y is

and Z² is alkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein n¹ is 1.

In certain embodiments, the present invention relates to theaforementioned compound, wherein t is 0 or 1.

In certain embodiments, the present invention relates to theaforementioned compound, wherein x is 1.

In certain embodiments, the present invention relates to theaforementioned compound, wherein the n² is 17, 18, 19, 20, 21, 22, or23.

In certain embodiments, the present invention relates to theaforementioned compound, wherein n² is 19, 20, or 21.

In certain embodiments, the present invention relates to theaforementioned compound, wherein n² is 20.

In certain embodiments, the present invention relates to theaforementioned compound, wherein n² is 20, and said first strand andsaid second strand are hybridized so that there is one unhybridizednucleoside on said first strand and said second strand.

In certain embodiments, the present invention relates to theaforementioned compound, wherein n is 20, and said first strand and saidsecond strand are hybridized so that there are two unhybridizednucleosides on said first strand and said second strand.

In certain embodiments, the present invention relates to theaforementioned compound, wherein n² is 20 for said first strand, and n²is 22 for said second strand.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R¹ is H.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R² and R³ represent independently foreach occurrence OH, F, —Oalkyl, —Oallyl, —Oalkylamine, or—O-A⁶-[A⁷-(A⁵)_(w)]_(y).

In certain embodiments, the present invention relates to theaforementioned compound, wherein R² and R³ represent independently foreach occurrence OH, F, —Oalkyl, —Oallyl, or —Oalkylamine.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R² and R³ represent independently foreach occurrence OH, F, —Oalkyl, —N(R¹⁹)₂, or —O— A⁶-[A⁷-(A⁵)_(w)]_(y).

In certain embodiments, the present invention relates to theaforementioned compound, wherein R³ represents independently for eachoccurrence H, OH, F, —OCH₃, —O(CH₂)₂OR¹⁹, —O(CH₂)₂SR¹⁹, —O(CH₂)₂N(R¹⁹)₂,—OCH₂C(O)N(H)CH₃, —NH₂, —N(CH₃)₂, —N(H)CH₃, —SCH₃, —O(CH₂)₂OCH₃,—O(CH₂)₂SCH₃, —O(CH₂)₂O(CH₂)₂N(CH₃)₂, or —O(CH₂)₂ON(CH₃)₂.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R³ represents independently for eachoccurrence —NH₂, —N(CH₃)₂, or —N(H)CH₃.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R⁴ represents independently for eachoccurrence H, OH, F, —OCH₃, —O(CH₂)₂OR¹⁹, —O(CH₂)₂SR¹⁹, —O(CH₂)₂N(R¹⁹)₂,—OCH₂C(O)N(H)CH₃, —NH₂, —N(CH₃)₂, —N(H)CH₃, —SCH₃, —O(CH₂)₂OCH₃,—O(CH₂)₂SCH₃, —O(CH₂)₂O(CH₂)₂N(CH₃)₂, or —O(CH₂)₂ON(CH₃)₂.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R⁴ represent independently for eachoccurrence OH, F, —Oalkyl, —Oallyl, or —Oalkylamine.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R⁴ represents independently for eachoccurrence —NH₂, —N(CH₃)₂, or —N(H)CH₃.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R³ and R⁴ represent independently foreach occurrence —NH₂, —N(CH₃)₂, or —N(H)CH₃.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R⁵ is H.

In certain embodiments, the present invention relates to theaforementioned compound, wherein Z² represents independently for eachoccurrence methyl, —OM, —Oalkyl, —Oaryl, —Oaralkyl, —SM, —Salkyl,—Saryl, —Saralkyl, —[C(R⁵)₂]_(m)N(R⁶)₂, —N(R¹⁰)R¹¹, or—N(R¹⁹)(C(R¹⁹)₂)_(m)N(R¹⁹)₂.

In certain embodiments, the present invention relates to theaforementioned compound, wherein Z³ is O.

In certain embodiments, the present invention relates to theaforementioned compound, wherein Z⁴ represents independently for eachoccurrence —OM, —Oalkyl, —Oaryl, or —Oaralkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein Z⁴ represents independently for eachoccurrence methyl, —OM, —Oalkyl, —Oaryl, —Oaralkyl, —SM, —Salkyl,—Saryl, —Saralkyl, —[C(R⁵)₂]_(m)N(R⁶)₂, —N(R¹⁰)R¹¹, or—N(R¹⁹)(C(R¹⁹)₂)_(m)N(R¹⁹)₂.

In certain embodiments, the present invention relates to theaforementioned oligonucleotide, wherein A² represents independently foreach occurrence:

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁴ represents independently for eachoccurrence

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁴ represents independently for eachoccurrence

and A⁵ represents independently for each occurrence

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁴ represents independently for eachoccurrence

and A⁵ represents independently for each occurrence

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ occurrs at least once.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ occurrs at least five times.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ occurrs at least ten times.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ occurrs only in said first strand.

In certain embodiments, the present invention relates to theaforementioned compound, wherein said first strand and said secondstrand each contain at least one occurrence of A⁵.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is —(C(R)₂)_(m)-A⁹⁹, wherein A⁹⁹ isoptionally substituted phenyl, naphthyl, anthracenyl, phenanthrenyl,pyrenyl, pyridinyl, quinolinyl, acridinyl, phenathridinyl, pyrazinyl,pyrimidinyl, pyridazinyl, quinoxalinyl, quinazolinyl,1,7-phenanthrolinyl, indolyl, thianaphthenyl, benzoxazolyl,benzofuranyl, 1,2-benzisoxazolyl, benzimidazolyl, pyrrolyl, thiophenyl,isoxazolyl, pyrazolyl, thiazolyl, imidazolyl, or tetrazolyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula III:

wherein

R^(1-III), R^(2-III), and R^(3-III) represent independently for eachoccurrence H, halogen, amino, hydroxyl, alkyl, alkoxyl, aminoalkyl,alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, thiol,thioalkyl, silyl, nitro, nitrile, acyl, acylamino, —COR, or —CO₂R.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula III, andR^(1-III) is alkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula III, andR^(1-III) is methyl, ethyl, propyl, isopropyl, butyl, sec-butyl,isobutyl, or tert-butyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula III, andR^(1-III) is methyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula III, andR^(2-III) is H.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula III, andR^(3-III) is alkoxyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula III, andR^(3-III) is methoxy, ethoxy, propoxy, isopropoxy, butoxy, sec-butoxy,isobutoxy, or tert-butoxy.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula III, andR^(3-III) is methoxy.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula III,R^(1-III) is methyl, R^(2-III) is H, and R^(3-III) is methoxy.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula IV:

IV

wherein

R^(1-IV), R^(2-IV), and R^(3-IV) represent independently for eachoccurrence H, halogen, amino, hydroxyl, alkyl, alkoxyl, aminoalkyl,alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, thiol,thioalkyl, silyl, nitro, nitrile, acyl, acylamino, —COR, or —CO₂R.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula IV, andR^(1-IV) is alkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula IV, andR^(1-IV) is methyl, ethyl, propyl, isopropyl, butyl, sec-butyl,isobutyl, or tert-butyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula IV, andR^(1-IV) is methyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula IV, andR^(2-IV) is H.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula IV, andR^(3-IV) is alkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula IV, andR^(3-IV) is methyl, ethyl, propyl, isopropyl, butyl, sec-butyl,isobutyl, tert-butyl, pentyl, hexyl, or heptyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula IV, andR^(3-IV) is isobutyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula IV,R^(1-IV) is methyl, R^(2-IV) is H, and R^(3-IV) is isobutyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula IV, and R²represents independently for each occurrence H, OH, F, or —Oalkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula IV, and R³and R⁴ represent independently for each occurrence —NH₂, —N(H)CH₃, or—N(CH₃)₂.

In certain embodiments, the present invention relates to theaforementioned compound, further provided that at least ten instances ofY are not

In certain embodiments, the present invention relates to theaforementioned compound, further provided that at least one instance ofY is not

In certain embodiments, the present invention relates to theaforementioned compound, further provided that at least ten instances ofY are not

In certain embodiments, the present invention relates to theaforementioned compound, further provided that at least one instance ofY is not

In certain embodiments, the present invention relates to theaforementioned compound, further provided that at least ten instances ofY are not

In certain embodiments, the present invention relates to theaforementioned compound, further provided that at least one instance ofY is not

In certain embodiments, the present invention relates to theaforementioned compound, further provided that at least ten instances ofY are not

Another aspect of the present invention relates to a single-strandedoligonucleotide represented by formula V:

wherein

X¹ is H, —P(O)(OM)₂, —P(O)(OM)-O—P(O)(OM)₂, —P(O)(Oalkyl)₂,—P(O)(Oalkyl)-O—P(O)(Oalkyl)₂, or -A⁶-[A⁷-(A⁵)_(w)]_(y);

M represents independently for each occurrence an alkali metal or atransition metal with an overall charge of +1;

R¹ and R⁵ represent independently for each occurrence H, alkyl, orhalogen;

R² and R³ represent independently for each occurrence H, OH, F, —Oalkyl,—Oallyl, —O(C(R¹⁹)₂)_(k)OR¹⁹, —O(C(R¹⁹)₂)_(k)SR¹⁹,—O(C(R¹⁹)₂)_(k)N(R¹⁹)₂, —O(C(R¹⁹)₂)_(k)C(O)N(R¹⁹)₂, —N(R¹⁹)₂,—S(C₁-C₆)alkyl, —O(C(R¹⁹)₂)_(k)O(C₁-C₆)alkyl,—O(C(R¹⁹)₂)_(k)S(C₁-C₆)alkyl,—O(C(R¹⁹)₂)_(k)O(C(R¹⁹)₂)_(k)N((C₁-C₆)alkyl)₂,—O(C(R¹⁹)₂)_(k)ON((C₁-C₆)alkyl)₂, or —O-A⁶-[A⁷-(A⁵)_(w)]_(y);

R⁴ represents independently for each occurrence H, OH, F, —Oalkyl,—Oallyl, —O(C(R¹⁹)₂)_(k)OR¹⁹, —O(C(R¹⁹)₂)_(k)SR¹⁹,—O(C(R¹⁹)₂)_(k)N(R¹⁹)₂, —O(C(R¹⁹)₂)_(k)C(O)N(R¹⁹)₂, —N(R¹⁹)₂,—S(C₁-C₆)alkyl, —O(C(R¹⁹)₂)_(k)O(C₁-C₆)alkyl,—O(C(R¹⁹)₂)_(k)S(C₁-C₆)alkyl,O(C(R¹⁹)₂)_(k)O(C(R¹⁹)₂)_(k)N((C₁-C₆)alkyl)₂, or—O(C(R¹⁹)₂)_(k)ON((C₁-C₆)alkyl)₂;

R⁶, R⁷, and R⁹ represent independently for each occurrence H, alkyl,aryl, or aralkyl;

R⁸ represents independently for each occurrence alkyl, aryl, or aralkyl;

k represents independently for each occurrence 1, 2, 3, or 4;

n¹ is 1, 2, or 3;

n² is an integer in the range of about 15-28, inclusive;

w represents independently for each occurrence 1, 2, or 3 in accord withthe rules of valence;

x represents independently for each occurrence 0, 1, 2, or 3;

y represents independently for each occurrence 1, 2, 3, 4, or 5 inaccord with the rules of valence;

A¹ represents independently for each occurrence:

A² represents independently for each occurrence:

A³ represents independently for each occurrence

A⁴ represents independently for each occurrence a bond, alkyl diradical,heteroalkyl diradical, alkenyl diradical, alkynyl diradical,alkylalkynyl diradical, aminoalkyl diradical, thioether, —C(O)—, —S(O)—,—S(O)₂—, B¹C(R)₂B², B¹C(R)(B²)₂, B¹C(B²)₃, B¹N(R)(B²), B¹N(B²)₂, or hasthe formula:

B¹ is a bond between A³ and A⁴;

B² is a bond between A⁴ and A⁵;

R represents independently for each occurrence hydrogen or alkyl;

m represents independently for each occurrence 1, 2, 3, 4, 5, 6, 7, or8;

m¹ represents independently for each occurrence 0, 1, 2, 3, 4, 5, 6, 7,or 8;

Y represents independently for each occurrence an alkyl diradical,cycloalkyl diradical, heteroalkyl diradical, heterocycloalkyl diradical,alkenyl diradical, alkynyl diradical, aryl diradical, heteroaryldiradical, aralkyl diradical, heteroaralkyl diradical,—X²C(O)X²[C(R⁵)₂]_(v)X²—, —X²C(NR⁶)X²[C(R⁵)₂]_(v)X²—,—X²C(S)X²[C(R⁵)₂]_(v)X²—, —X²C(O)X²[C(R⁵)₂]_(v)X²C(O)X²—,

[C(R⁵)₂]_(t)N(R⁶)O[C(R⁵)₂]_(t)—, —[C(R⁵)₂]_(t)N(R⁶)N(R⁶)O[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)N(R⁷)C(O)[C(R⁵)₂]_(t)—, —[C(R⁵)₂]_(t)N(R)CO₂[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)N(R⁷)C(S)[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)N(R⁷)C(S)O[C(R⁵)₂]_(t)—, —[C(R⁵)₂]_(t)OC(O)S[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)SN(R⁷)CO₂[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)OSi(R⁸)₂O[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)OSO₂N(R⁷)[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)N(R⁷)SO₂N(R⁷)[C(R⁵)₂]—,—[C(R⁵)₂]_(t)SO₂N(morpholino)-[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)SO₂N(R⁷)[C(R⁵)₂]_(t)—, —[C(R¹)₂]_(t)S[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)OSO₂[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)S[C(R⁵)₂]_(y)O[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)O[C(R⁵)₂]_(y)O[C(R⁵)₂]_(t)—, —[C(R⁵)₂]_(t)O[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)N(R⁷)[C(R⁵)₂]_(t)—, —[C(R⁵)₂]_(t)C═NO[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)C(O)C(R⁵)═C(R⁵)[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)C(R⁵)═C(R⁵)[C(R⁵)₂]_(t)—, or[C(R⁵)₂]_(t)X²C(O)X²[C(R⁵)₂]_(t)—;

X² represents independently for each occurrence a bond, O, or N(R⁶);

Z¹ represents independently for each occurrence O, S, or N(R⁸);

Z² represents independently for each occurrence alkyl, aryl, aralkyl,B(R⁹)₃, —OM, —Oalkyl, —Oaryl, —Oaralkyl, —SM, —Salkyl, —Saryl,—Saralkyl, —[C(R⁵)₂]_(m)N(R⁶)₂, —N(R¹⁰)R¹¹, —N(R¹⁹)(C(R¹⁹)₂)_(m)N(R¹⁹)₂,—N(R⁷)C(O)R⁸, H, —OC(O)R⁸, —CO₂R⁸, F, Se, —SeR⁸, —(C(R¹⁹)₂)_(m)OR¹⁹,—(C(R¹⁹)₂)_(m)SR¹⁹, N(R¹⁹)(C(R¹⁹)₂)_(m)OR¹⁹, N(R¹⁹)(C(R¹⁹)₂)_(m)SR¹⁹,—N(R¹⁹)(C(R¹⁹)₂)_(m)N(R¹⁹)C(O)alkyl, —(C(R¹⁹)₂)_(m)N(R¹⁹)C(O)alkyl, or-A⁸-[A⁹-(A⁵)_(w)]_(y);

R¹⁰ and R¹¹ are independently H, alkyl, or aryl; or R¹⁰ and R¹¹ takentogether form a 3-, 4-, 5-, 6-, or 7-member ring;

R¹² represents independently for each occurrence H, alkyl or—NHCH₂CH═CH₂;

t represents independently for each occurrence 0, 1, 2, 3, or 4;

v represents independently for each occurrence 0, 1, 2, 3, 4, 5, 6, 7,or 8;

A⁵ represents independently for each occurrence aryl, aralkyl, or theradical of a steroid, bile acid, lipid, folic acid, pyridoxal, B12,riboflavin, biotin, polycyclic compound, crown ether, intercalator,cleaver molecule, protein-binding agent, carbohydrate, or an optionallysubstituted saturated 5-membered ring;

A⁶ represents independently for each occurrence a bond, alkyl diradical,heteroalkyl diradical, alkenyl diradical, aminoalkyl, —C(O)—, —S(O)—,—S(O)₂—, or is represented by formula:

Z³ represents independently for each occurrence O or S;

Z⁴ represents independently for each occurrence —OM, —Oalkyl, —Oaryl,—Oaralkyl, —SM, —Salkyl, —Saryl, —Saralkyl, —N(R¹⁰)R¹¹,—[C(R⁵)₂]_(m)N(R⁶)₂, —N(R¹⁹)(C(R¹⁹)₂)_(m)N(R¹⁹)₂, (C(R¹⁹)₂)_(m)OR¹⁹,—(C(R¹⁹)₂)_(m)SR¹⁹, —N(R¹⁹)(C(R¹⁹)₂)_(m)OR¹⁹, —N(R¹⁹)(C(R¹⁹)₂)_(m)SR⁹,—N(R¹⁹)(C(R¹⁹)₂)_(m)N(R¹⁹)C(O)alkyl, —(C(R¹⁹)₂)_(m)N(R¹⁹)C(O)alkyl,aryl, or alkyl;

R¹³ represent independently for each occurrence H, alkyl, cycloalkyl,heteroalkyl, aryl, aralkyl, acyl, silyl, or B³;

R¹⁴ represents independently for each occurrence alkyl, aryl, aralkyl,acyl, or silyl;

R¹⁵ represents independently for each occurrence hydrogen, alkyl, aryl,aralkyl, acyl, alkylsulfonyl, alkylsulfoxide, arylsulfonyl,arylsulfoxide, or silyl;

R¹⁶ represents independently for each occurrence cycloalkyl,heterocycloalkyl, aryl, or heteroaryl;

B³ is a bond between A⁶ and A⁷;

B⁴ is a bond between A⁶ and O;

n³ represents independently for each occurrence an integer in the rangeof 1-15, inclusive;

n⁴ represents independently for each occurrence 1, 2, 3, 4, or 5 inaccord with the rules of valence;

A⁷ represents independently for each occurrence a bond, alkyl diradical,heteroalkyl diradical, —C(O)—, —S(O)—, —S(O)₂—, B³C(R)₂B⁵, B³C(R)(B⁵)₂,B³C(B⁵)₃, B³N(R)(B⁵), B³N(B⁵)₂, or has the formula:

p represents independently for each occurrence 1, 2, 3, or 4;

B⁵ is a bond between A⁵ and A⁷;

A⁸ is a bond, alkyl diradical, heteroalkyl diradical, alkenyl diradical,aminoalkyl, or is represented by formula:

R¹⁷ represent independently for each occurrence H, alkyl, cycloalkyl,heteroalkyl, aryl, aralkyl, acyl, silyl, or B⁶;

R¹⁸ represents independently for each occurrence H, halogen, alkyl,alkoxyl, —N(R)₂, —CN, —[C(R⁵)₂]_(v)C(R⁵)═C(R⁵)₂;

R¹⁹ represents independently for each occurrence H or alkyl;

B⁶ is a bond between A⁸ and A⁹;

B⁷ is a bond between A⁸ and P;

A⁹ is a bond, alkyl diradical, heteroalkyl diradical, —C(O)—, —S(O)—,—S(O)₂—, B⁶C(R)₂B⁸, B⁶C(R)(B⁸)₂, B⁶C(B⁸)₃, B⁶N(R)(B⁸), B⁶N(B⁸)₂, or hasthe formula:

B⁸ is a bond between A⁵ and A⁹; and provided that at least one instanceof Y is not

In certain embodiments, the present invention relates to theaforementioned compound, wherein A¹ represents independently for eachoccurrence:

In certain embodiments, the present invention relates to theaforementioned compound, wherein Y is

In certain embodiments, the present invention relates to theaforementioned compound, wherein Y is

and Z² represents independently for each occurrence alkyl, aryl,aralkyl, B(R)₃, —OM, —Oalkyl, —Oaryl, or —Oaralkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein Y is

and Z² represents independently for each occurrence alkyl or —OM.

In certain embodiments, the present invention relates to theaforementioned compound, wherein Y is

and Z² represents independently for each occurrence methyl, ethyl,propyl, isopropyl, or —OM.

In certain embodiments, the present invention relates to theaforementioned compound, wherein Y is

and Z² represents independently for each occurrence methyl or —OM.

In certain embodiments, the present invention relates to theaforementioned compound, wherein Y is

and there are at least two instances when Z² is alkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein Y is

and there are at least five instances when Z² is alkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein Y is

and there are at least seven instances when Z² is alkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein Y is

and there are at least ten instances when Z² is alkyl:

In certain embodiments, the present invention relates to theaforementioned compound, wherein in the linkage between the firstnucleoside and second nucleoside at the terminus of said first strand, Yis

and Z² is alkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein in the linkage between the first andsecond nucleoside at the 3′-terminus of said first strand, Y is

and Z² is alkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein in the linkage between the first andsecond nucleoside at the 3′-terminus of said first strand and saidsecond strand, Y is

and Z² is alkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein n, is 1.

In certain embodiments, the present invention relates to theaforementioned compound, wherein t is 0 or 1.

In certain embodiments, the present invention relates to theaforementioned compound, wherein x is 1.

In certain embodiments, the present invention relates to theaforementioned compound, wherein the n² is 17, 18, 19, 20, 21, 22, or23.

In certain embodiments, the present invention relates to theaforementioned compound, wherein n² is 19, 20, or 21.

In certain embodiments, the present invention relates to theaforementioned compound, wherein n² is 20.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R¹ is H.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R² and R³ represent independently foreach occurrence OH, F, —Oalkyl, —Oallyl, —Oalkylamine, or—O-A⁶-[A⁷-(A⁵)_(w)]_(y).

In certain embodiments, the present invention relates to theaforementioned compound, wherein R² and R³ represent independently foreach occurrence OH, F, —Oalkyl, —Oallyl, or —Oalkylamine.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R² and R³ represent independently foreach occurrence OH, F, —Oalkyl, —Oallyl, —Oalkylamine, —N(R¹⁹)₂, or—O-A⁶-[A⁷-(A⁵)_(w)]_(y).

In certain embodiments, the present invention relates to theaforementioned compound, wherein R² and R³ represent independently foreach occurrence OH, F, —Oalkyl, —Oallyl, —Oalkylamine, or —N(R¹⁹)₂.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R³ represents independently for eachoccurrence H, OH, F, —OCH₃, —O(CH₂)₂OR¹⁹, —O(CH₂)₂SR¹⁹, —O(CH₂)₂N(R¹⁹)₂,—OCH₂C(O)N(H)CH₃, —NH₂, —N(CH₃)₂, —N(H)CH₃, —SCH₃, —O(CH₂)₂OCH₃,—O(CH₂)₂SCH₃, —O(CH₂)₂O(CH₂)₂N(CH₃)₂, or —O(CH₂)₂ON(CH₃)₂.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R³ represents independently for eachoccurrence —NH₂, —N(CH₃)₂, or —N(H)CH₃.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R⁴ represents independently for eachoccurrence H, OH, F, —OCH₃, —O(CH₂)₂OR¹⁹, —O(CH₂)₂SR¹⁹, —O(CH₂)₂N(R¹⁹)₂,—OCH₂C(O)N(H)CH₃, —NH₂, —N(CH₃)₂, —N(H)CH₃, —SCH₃, —O(CH₂)₂OCH₃,—O(CH₂)₂SCH₃, —O(CH₂)₂O(CH₂)₂N(CH₃)₂, or —O(CH₂)₂ON(CH₃)₂.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R⁴ represents independently for eachoccurrence OH, F, —Oalkyl, —Oallyl, or —Oalkylamine.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R⁴ represents independently for eachoccurrence —NH₂, —N(H)CH₃, or —N(CH₃)₂.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R³ and R⁴ represent independently OH,—Oalkyl, or —Oallyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R³ and R⁴ represent independently —NH₂,—N(H)CH₃, or —N(CH₃)₂.

In certain embodiments, the present invention relates to theaforementioned compound, wherein at least two instances of R⁴ are OH.

In certain embodiments, the present invention relates to theaforementioned compound, wherein at least five instances of R⁴ are OH.

In certain embodiments, the present invention relates to theaforementioned compound, wherein at least ten instances of R⁴ are OH.

In certain embodiments, the present invention relates to theaforementioned compound, wherein at least fifteen instances of R⁴ areOH.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R⁵ is H.

In certain embodiments, the present invention relates to theaforementioned compound, wherein Z² represents independently for eachoccurrence methyl, —OM, —Oalkyl, —Oaryl, —Oaralkyl, —SM, —Salkyl,—Saryl, —Saralkyl, —[C(R⁵)₂]_(m)N(R⁶)₂, —N(R¹⁰)R¹¹, or—N(R¹⁹)(C(R¹⁹)₂)_(m)N(R¹⁹)₂.

In certain embodiments, the present invention relates to theaforementioned compound, wherein Z³ is O.

In certain embodiments, the present invention relates to theaforementioned compound, wherein Z⁴ represents independently for eachoccurrence —OM, —Oalkyl, —Oaryl, or —Oaralkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein Z⁴ represents independently for eachoccurrence methyl, —OM, —Oalkyl, —Oaryl, —Oaralkyl, —SM, —Salkyl,—Saryl, —Saralkyl, —[C(R⁵)₂]_(m)N(R⁶)₂, —N(R¹⁰)R¹¹, or—N(R¹⁹)(C(R¹⁹)₂)_(m)N(R¹⁹)₂.

In certain embodiments, the present invention relates to theaforementioned oligonucleotide, wherein A² represents independently foreach occurrence:

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁴ represents independently for eachoccurrence

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁴ represents independently for eachoccurrence

and A⁵ represents independently for each occurrence

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁴ represents independently for eachoccurrence

and A⁵ represents independently for each occurrence

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ occurrs at least once.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ occurrs at least five times.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is (C(R)₂)_(m)-A⁹⁹, wherein A⁹⁹ isoptionally substituted phenyl, naphthyl, anthracenyl, phenanthrenyl,pyrenyl, pyridinyl, quinolinyl, acridinyl, phenathridinyl, pyrazinyl,pyrimidinyl, pyridazinyl, quinoxalinyl, quinazolinyl,1,7-phenanthrolinyl, indolyl, thianaphthenyl, benzoxazolyl,benzofuranyl, 1,2-benzisoxazolyl, benzimidazolyl, pyrrolyl, thiophenyl,isoxazolyl, pyrazolyl, thiazolyl, imidazolyl, or tetrazolyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula VI:

wherein

R^(1-VI), R^(2-VI), and R^(3-VI) represent independently for eachoccurrence H, halogen, amino, hydroxyl, alkyl, alkoxyl, aminoalkyl,alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, thiol,thioalkyl, silyl, nitro, nitrile, acyl, acylamino, —COR, or —CO₂R.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula VI, andR^(1-VI) is alkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula VI, andR^(1-VI) is methyl, ethyl, propyl, isopropyl, butyl, sec-butyl,isobutyl, or tert-butyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula VI, andR^(1-VI) is methyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula VI, andR^(2-VI) is H.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula VI, andR^(3-VI) is alkoxyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula VI, andR^(3-VI) is methoxy, ethoxy, propoxy, isopropoxy, butoxy, sec-butoxy,isobutoxy, or tert-butoxy.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula VI, andR^(3-VI) is methoxy.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula VI,R^(1-VI) is methyl, R^(2-VI) is H, and R^(3-VI) is methoxy.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula VII:

wherein

R^(1-VII), R^(2-VII), and R^(3-VII) represent independently for eachoccurrence H, halogen, amino, hydroxyl, alkyl, alkoxyl, aminoalkyl,alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, thiol,thioalkyl, silyl, nitro, nitrile, acyl, acylamino, —COR, or —CO₂R.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula VII, andR^(1-VII) is alkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula VII, andR^(1-VII) is methyl, ethyl, propyl, isopropyl, butyl, sec-butyl,isobutyl, or tert-butyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula VII, andR^(1-VII) is methyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula VII, andR^(2-VII) is H.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula VII, andR^(3-VII) is alkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula VII, andR^(3-VII) is methyl, ethyl, propyl, isopropyl, butyl, sec-butyl,isobutyl, tert-butyl, pentyl, hexyl, or heptyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula VII, andR^(3-VII) is isobutyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula VII,R^(1-VII) is methyl, R^(2-VII) is H, and R^(3-VII) is isobutyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is represented by formula VII, andR² represents independently for each occurrence H, OH, F, or —Oalkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R³ and R⁴ represent independently foreach occurrence —NH₂, —N(H)CH₃, or —N(CH₃)₂.

In certain embodiments, the present invention relates to theaforementioned compound, further provided that at least ten instances ofY are not

In certain embodiments, the present invention relates to theaforementioned compound, further provided that at least one instance ofY is not

In certain embodiments, the present invention relates to theaforementioned compound, further provided that at least ten instances ofY are not

In certain embodiments, the present invention relates to theaforementioned compound, further provided that at least one instance ofY is not

In certain embodiments, the present invention relates to theaforementioned compound, further provided that at least ten instances ofY are not

In certain embodiments, the present invention relates to theaforementioned compound, further provided that at least one instance ofY is not

In certain embodiments, the present invention relates to theaforementioned compound, further provided that at least ten instances ofY are not

Methods of the Invention

One aspect of the present invention relates to a method of treating apatient suffering from a malady selected from the group consisting ofunwanted cell proliferation, arthritis, retinal neovascularization,viral infection, bacterial infection, amoebic infection, parasiticinfection, fungal infection, unwanted immune response, asthma, lupus,multiple sclerosis, diabetes, acute pain, chronic pain, neurologicaldisease, and a disorder characterized by loss of heterozygosity;comprising the step of:

administering to a patient in need thereof a therapeutically effectiveamount of an oligonucleotide, wherein said oligonucleotide is asingle-stranded oligonucleotide represented by formula V as describedabove, or said oligonucleotide is a double-stranded oligonucleotidecomprising a first strand and a second strand, wherein said first strandand said second are represented independently by formula II as describedabove.

In certain embodiments, the present invention relates to theaforementioned method, wherein said malady is unwanted cellproliferation.

In certain embodiments, the present invention relates to theaforementioned method, wherein said malady is testicular cancer, lungcancer, breast cancer, colon cancer, squamous cell carcinoma, pancreaticcancer, leukemia, melanoma, Burkitt's lymphoma, neuroblastoma, ovariancancer, prostate cancer, skin cancer, non-Hodgkin lymphoma, esophagealcancer, cervical cancer, basal cell carcinoma, adenocarcinoma carcinoma,hepatocellular carcinoma, colorectal adenocarcinoma, liver cancer, malebreast carcinoma, adenocarcinomas of the esophagus, adenocarcinomas ofthe stomach, adenocarcinomas of the colon, adenocarcinomas of therectum, gall bladder cancer, hamartomas, gliomas, endometrial cancer,acute leukemia, chronic leukemia, childhood acute leukemia, EwingSarcoma, Myxoid liposarcoma, brain cancer, or tumors of epithelialorigin.

In certain embodiments, the present invention relates to theaforementioned method, wherein said malady is rheumatoid arthritis orretinal neovascularization.

In certain embodiments, the present invention relates to theaforementioned method, wherein said malady is a viral infection.

In certain embodiments, the present invention relates to theaforementioned method, wherein said malady is a disorder mediated byHuman Papilloma Virus, Human Immunodeficiency Virus, Hepatitis A Virus,Hepatitis B Virus, Hepatitis C Virus, Hepatitis D Virus, Hepatitis EVirus, Hepatitis F Virus, Hepatitis G Virus, Hepatitis H Virus,Respiratory Syncytial Virus, Herpes Simplex Virus, herpesCytomegalovirus, herpes Epstein Barr Virus, a Kaposi'sSarcoma-associated Herpes Virus, JC Virus, myxovirus, rhinovirus,coronavirus, West Nile Virus, St. Louis Encephalitis, Tick-borneencephalitis virus gene, Murray Valley encephalitis virus gene, denguevirus gene, Simian Virus 40, Human T Cell Lymphotropic Virus, aMoloney-Murine Leukemia Virus, encephalomyocarditis virus, measlesvirus, Vericella zoster virus, adenovirus, yellow fever virus,poliovirus, or poxvirus.

In certain embodiments, the present invention relates to theaforementioned method, wherein said malady is a bacterial infection,amoebic infection, parasitic infection, or fungal infection.

In certain embodiments, the present invention relates to theaforementioned method, wherein said malady is a disorder mediated byplasmodium, Mycobacterium ulcerans, Mycobacterium tuberculosis,Mycobacterium leprae, Staphylococcus aureus, Streptococcus pneumoniae,Streptococcus pyogenes, Chlamydia pneumoniae, or Mycoplasma pneumoniae.

In certain embodiments, the present invention relates to theaforementioned method, wherein said malady is an unwanted immuneresponse, asthma, lupus, multiple sclerosis, or diabetes.

In certain embodiments, the present invention relates to theaforementioned method, wherein said malady is an ischemia, reperfusioninjury, response to a transplantated organ or tissue, restenosis, orInflammatory Bowel Disease.

In certain embodiments, the present invention relates to theaforementioned method, wherein said malady is acute pain or chronicpain.

In certain embodiments, the present invention relates to theaforementioned method, wherein said malady is a neurological disease.

In certain embodiments, the present invention relates to theaforementioned method, wherein said malady is Alzheimer Disease,Parkinson Disease, or a neurodegenerative trinucleotide repeat disorder.

In certain embodiments, the present invention relates to theaforementioned method, wherein said malady is a disorder characterizedby loss of heterozygosity.

In certain embodiments, the present invention relates to theaforementioned method, wherein said oligonucleotide is a double-strandedoligonucleotide comprising a first strand and a second strand, whereinsaid first strand and said second are represented independently byformula II as described above.

Another aspect of the present invention relates to a method ofgene-silencing, comprising the steps of:

administering a therapeutically effective amount of an oligonucleotideto a mammalian cell to silence a gene promoting unwanted cellproliferation, growth factor gene, growth factor receptor gene, a kinasegene, a gene encoding a G protein superfamily molecule, a gene encodinga transcription factor, a gene which mediates angiogenesis, a viral geneof a cellular gene which mediates viral function, a gene of a bacterialpathogen, a gene of an amoebic pathogen, a gene of a parasitic pathogen,a gene of a fungal pathogen, a gene which mediates an unwanted immuneresponse, a gene which mediates the processing of pain, a gene whichmediates a neurological disease, an allene gene found in cellscharacterized by loss of heterozygosity, or one allege gene of apolymorphic gene; wherein said oligonucleotide is a single-strandedoligonucleotide represented by formula V as described above, or saidoligonucleotide is a double-stranded oligonucleotide comprising a firststrand and a second strand represented by formula II as described above.

In certain embodiments, the present invention relates to theaforementioned method, wherein said oligonucleotide is a double-strandedoligonucleotide comprising a first strand and a second strand, whereinsaid first strand and said second are represented independently byformula II as described above.

Another aspect of the present invention relates to a method ofgene-silencing, comprising the steps of:

administering a therapeutically effective amount of an oligonucleotideto a mammalian cell to silence a PDGF beta gene, Erb-B gene, Src gene,CRK gene, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1/2gene, PCNA(p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, Cyclin Dgene, VEGF gene, EGFR gene, Cyclin A gene, Cyclin E gene, WNT-1 gene,beta-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivingene, Her2/Neu gene, topoisomerase I gene, topoisomerase II alpha gene,mutations in the p73 gene, mutations in the p21(WAF1/CIP1) gene,mutations in the p27(KIP1) gene, mutations in the PPMID gene, mutationsin the RAS gene, mutations in the caveolin I gene, mutations in the MIBI gene, mutations in the MTAI gene, mutations in the M68 gene, mutationsin tumor suppressor genes, mutations in the p53 tumor suppressor gene,mutations in the p53 family member DN-p63, mutations in the pRb tumorsuppressor gene, mutations in the APC1 tumor suppressor gene, mutationsin the BRCA1 tumor suppressor gene, mutations in the PTEN tumorsuppressor gene, mLL fusion gene, BCR/ABL fusion gene, TEL/AML1 fusiongene, EWS/FLI1 fusion gene, TLS/FUS1 fusion gene, PAX3/FKHR fusion gene,AML1/ETO fusion gene, alpha v-integrin gene, Flt-1 receptor gene,tubulin gene, Human Papilloma Virus gene, a gene required for HumanPapilloma Virus replication, Human immunodeficiency Virus gene, a generequired for Human Immunodeficiency Virus replication, Hepatitis A Virusgene, a gene required for Hepatitis A Virus replication, Hepatitis BVirus gene, a gene required for Hepatitis B Virus replication, HepatitisC Virus gene, a gene required for Hepatitis C Virus replication,Hepatitis D Virus gene, a gene required for Hepatitis D Virusreplication, Hepatitis E Virus gene, a gene required for Hepatitis EVirus replication, Hepatitis F Virus gene, a gene required for HepatitisF Virus replication, Hepatitis G Virus gene, a gene required forHepatitis G Virus replication, Hepatitis H Virus gene, a gene requiredfor Hepatitis H Virus replication, Respiratory Syncytial Virus gene, agene that is required for Respiratory Syncytial Virus replication,Herpes Simplex Virus gene, a gene that is required for Herpes SimplexVirus replication, herpes Cytomegalovirus gene, a gene that is requiredfor herpes Cytomegalovirus replication, herpes Epstein Barr Virus gene,a gene that is required for herpes Epstein Barr Virus replication,Kaposi's Sarcoma-associated Herpes Virus gene, a gene that is requiredfor Kaposi's Sarcoma-associated Herpes Virus replication, JC Virus gene,human gene that is required for JC Virus replication, myxovirus gene, agene that is required for myxovirus gene replication, rhinovirus gene, agene that is required for rhinovirus replication, coronavirus gene, agene that is required for coronavirus replication, West Nile Virus gene,a gene that is required for West Nile Virus replication, St. LouisEncephalitis gene, a gene that is required for St. Louis Encephalitisreplication, Tick-borne encephalitis virus gene, a gene that is requiredfor Tick-borne encephalitis virus replication, Murray Valleyencephalitis virus gene, a gene that is required for Murray Valleyencephalitis virus replication, dengue virus gene, a gene that isrequired for dengue virus gene replication, Simian Virus 40 gene, a genethat is required for Simian Virus 40 replication, Human T CellLymphotropic Virus gene, a gene that is required for Human T CellLymphotropic Virus replication, Moloney-Murine Leukemia Virus gene, agene that is required for Moloney-Murine Leukemia Virus replication,encephalomyocarditis virus gene, a gene that is required forencephalomyocarditis virus replication, measles virus gene, a gene thatis required for measles virus replication, Vericella zoster virus gene,a gene that is required for Vericella zoster virus replication,adenovirus gene, a gene that is required for adenovirus replication,yellow fever virus gene, a gene that is required for yellow fever virusreplication, poliovirus gene, a gene that is required for poliovirusreplication, poxvirus gene, a gene that is required for poxvirusreplication, plasmodium gene, a gene that is required for plasmodiumgene replication, Mycobacterium ulcerans gene, a gene that is requiredfor Mycobacterium ulcerans replication, Mycobacterium tuberculosis gene,a gene that is required for Mycobacterium tuberculosis replication,Mycobacterium leprae gene, a gene that is required for Mycobacteriumleprae replication, Staphylococcus aureus gene, a gene that is requiredfor Staphylococcus aureus replication, Streptococcus pneumoniae gene, agene that is required for Streptococcus pneumoniae replication,Streptococcus pyogenes gene, a gene that is required for Streptococcuspyogenes replication, Chlamydia pneumoniae gene, a gene that is requiredfor Chlamydia pneumoniae replication, Mycoplasma pneumoniae gene, a genethat is required for Mycoplasma pneumoniae replication, an integringene, a selectin gene, complement system gene, chemokine gene, chemokinereceptor gene, GCSF gene, Gro1 gene, Gro2 gene, Gro3 gene, PF4 gene, MIGgene, Pro-Platelet Basic Protein gene, MIP-1I gene, MIP-1J gene, RANTESgene, MCP-1 gene, MCP-2 gene, MCP-3 gene, CMBKR1 gene, CMBKR2 gene,CMBKR3 gene, CMBKR5v, AIF-1 gene, I-309 gene, a gene to a component ofan ion channel, a gene to a neurotransmitter receptor, a gene to aneurotransmitter ligand, amyloid-family gene, presenilin gene, HD gene,DRPLA gene, SCA1 gene, SCA2 gene, MJD1 gene, CACNL1A4 gene, SCA7 gene,SCA8 gene, allele gene found in LOH cells, or one allele gene of apolymorphic gene; wherein said oligonucleotide is a single-strandedoligonucleotide represented by formula V as described above, or saidoligonucleotide is a double-stranded oligonucleotide comprising a firststrand and a second strand represented by formula II as described above.

In certain embodiments, the present invention relates to theaforementioned method, wherein said oligonucleotide is a double-strandedoligonucleotide comprising a first strand and a second strand, whereinsaid first strand and said second are represented independently byformula II as described above.

Another aspect of the present invention relates to a method ofgene-silencing, comprising the steps of:

administering a therapeutically effective amount of an oligonucleotideto a mammal to silence a gene promoting unwanted cell proliferation,growth factor or growth factor receptor gene, a kinase gene, a geneencoding a G protein superfamily molecule, a gene encoding atranscription factor, a gene which mediates angiogenesis, a viral geneof a cellular gene which mediates viral function, a gene of a bacterialpathogen, a gene of an amoebic pathogen, a gene of a parasitic pathogen,a gene of a fungal pathogen, a gene which mediates an unwanted immuneresponse, a gene which mediates the processing of pain, a gene whichmediates a neurological disease, an allene gene found in cellscharacterized by loss of heterozygosity, or one allege gene of apolymorphic gene; wherein said oligonucleotide is a single-strandedoligonucleotide represented by formula V as described above, or saidoligonucleotide is a double-stranded oligonucleotide comprising a firststrand and a second strand represented by formula II as described above.

In certain embodiments, the present invention relates to theaforementioned method, wherein said oligonucleotide is a double-strandedoligonucleotide comprising a first strand and a second strand, whereinsaid first strand and said second are represented independently byformula II as described above.

Another aspect of the present invention relates to a method ofgene-silencing, comprising the steps of:

administering a therapeutically effective amount of an oligonucleotideto a mammal to silence a PDGF beta gene, Erb-B gene, Src gene, CRK gene,GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1/2 gene,PCNA(p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, Cyclin D gene,VEGF gene, EGFR gene, Cyclin A gene, Cyclin E gene, WNT-1 gene,beta-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivingene, Her2/Neu gene, topoisomerase I gene, topoisomerase II alpha gene,mutations in the p73 gene, mutations in the p21(WAF1/CIP1) gene,mutations in the p27(KIP1) gene, mutations in the PPMID gene, mutationsin the RAS gene, mutations in the caveolin I gene, mutations in the MIBI gene, mutations in the MTAI gene, mutations in the M68 gene, mutationsin tumor suppressor genes, mutations in the p53 tumor suppressor gene,mutations in the p53 family member DN-p63, mutations in the pRb tumorsuppressor gene, mutations in the APC1 tumor suppressor gene, mutationsin the BRCA1 tumor suppressor gene, mutations in the PTEN tumorsuppressor gene, mLL fusion gene, BCR/ABL fusion gene, TEL/AML1 fusiongene, EWS/FLI1 fusion gene, TLS/FUS1 fusion gene, PAX3/FKHR fusion gene,AML1/ETO fusion gene, alpha v-integrin gene, Flt-1 receptor gene,tubulin gene, Human Papilloma Virus gene, a gene required for HumanPapilloma Virus replication, Human Immunodeficiency Virus gene, a generequired for Human Immunodeficiency Virus replication, Hepatitis A Virusgene, a gene required for Hepatitis A Virus replication, Hepatitis BVirus gene, a gene required for Hepatitis B Virus replication, HepatitisC Virus gene, a gene required for Hepatitis C Virus replication,Hepatitis D Virus gene, a gene required for Hepatitis D Virusreplication, Hepatitis E Virus gene, a gene required for Hepatitis EVirus replication, Hepatitis F Virus gene, a gene required for HepatitisF Virus replication, Hepatitis G Virus gene, a gene required forHepatitis G Virus replication, Hepatitis H Virus gene, a gene requiredfor Hepatitis H Virus replication, Respiratory Syncytial Virus gene, agene that is required for Respiratory Syncytial Virus replication,Herpes Simplex Virus gene, a gene that is required for Herpes SimplexVirus replication, herpes Cytomegalovirus gene, a gene that is requiredfor herpes Cytomegalovirus replication, herpes Epstein Barr Virus gene,a gene that is required for herpes Epstein Barr Virus replication,Kaposi's Sarcoma-associated Herpes Virus gene, a gene that is requiredfor Kaposi's Sarcoma-associated Herpes Virus replication, JC Virus gene,human gene that is required for JC Virus replication, myxovirus gene, agene that is required for myxovirus gene replication, rhinovirus gene, agene that is required for rhinovirus replication, coronavirus gene, agene that is required for coronavirus replication, West Nile Virus gene,a gene that is required for West Nile Virus replication, St. LouisEncephalitis gene, a gene that is required for St. Louis Encephalitisreplication, Tick-borne encephalitis virus gene, a gene that is requiredfor Tick-borne encephalitis virus replication, Murray Valleyencephalitis virus gene, a gene that is required for Murray Valleyencephalitis virus replication, dengue virus gene, a gene that isrequired for dengue virus gene replication, Simian Virus 40 gene, a genethat is required for Simian Virus 40 replication, Human T CellLymphotropic Virus gene, a gene that is required for Human T CellLymphotropic Virus replication, Moloney-Murine Leukemia Virus gene, agene that is required for Moloney-Murine Leukemia Virus replication,encephalomyocarditis virus gene, a gene that is required forencephalomyocarditis virus replication, measles virus gene, a gene thatis required for measles virus replication, Vericella zoster virus gene,a gene that is required for Vericella zoster virus replication,adenovirus gene, a gene that is required for adenovirus replication,yellow fever virus gene, a gene that is required for yellow fever virusreplication, poliovirus gene, a gene that is required for poliovirusreplication, poxvirus gene, a gene that is required for poxvirusreplication, plasmodium gene, a gene that is required for plasmodiumgene replication, Mycobacterium ulcerans gene, a gene that is requiredfor Mycobacterium ulcerans replication, Mycobacterium tuberculosis gene,a gene that is required for Mycobacterium tuberculosis replication,Mycobacterium leprae gene, a gene that is required for Mycobacteriumleprae replication, Staphylococcus aureus gene, a gene that is requiredfor Staphylococcus aureus replication, Streptococcus pneumoniae gene, agene that is required for Streptococcus pneumoniae replication,Streptococcus pyogenes gene, a gene that is required for Streptococcuspyogenes replication, Chlamydia pneumoniae gene, a gene that is requiredfor Chlamydia pneumoniae replication, Mycoplasma pneumoniae gene, a genethat is required for Mycoplasma pneumoniae replication, an integringene, a selectin gene, complement system gene, chemokine gene, chemokinereceptor gene, GCSF gene, Gro1 gene, Gro2 gene, Gro3 gene, PF4 gene, MIGgene, Pro-Platelet Basic Protein gene, MIP-1I gene, MIP-1J gene, RANTESgene, MCP-1 gene, MCP-2 gene, MCP-3 gene, CMBKR1 gene, CMBKR2 gene,CMBKR3 gene, CMBKR5v, AIF-1 gene, 1-309 gene, a gene to a component ofan ion channel, a gene to a neurotransmitter receptor, a gene to aneurotransmitter ligand, amyloid-family gene, presenilin gene, HD gene,DRPLA gene, SCA1 gene, SCA2 gene, MJD1 gene, CACNL1A4 gene, SCA7 gene,SCA8 gene, allele gene found in LOH cells, or one allele gene of apolymorphic gene; wherein said oligonucleotide is a single-strandedoligonucleotide represented by formula V as described above, or saidoligonucleotide is a double-stranded oligonucleotide comprising a firststrand and a second strand represented by formula II as described above.

In certain embodiments, the present invention relates to theaforementioned method, wherein, said mammal is a primate, equine, canineor feline.

In certain embodiments, the present invention relates to theaforementioned method, wherein, said mammal is a human.

In certain embodiments, the present invention relates to theaforementioned method, wherein said oligonucleotide is a double-strandedoligonucleotide comprising a first strand and a second strand, whereinsaid first strand and said second are represented independently byformula II as described above.

Definitions

For convenience, certain terms employed in the specification, examples,and appended claims are collected here.

The term “silence” means to at least partially suppress. For example, incertain instances, the gene is suppressed by at least about 25%, 35%, or50% by administration of an oligonucleotide of the invention. In apreferred embodiment, the gene is suppressed by at least about 60%, 70%,or 80% by administration of an oligonucleotide of the invention. In amore preferred embodiment, the gene is suppressed by at least about 85%,90%, or 95% by administration of an oligonucleotide of the invention. Ina most preferred embodiment, the gene is suppressed by at least about98% or 99% by administration of an oligonucleotide of the invention.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are boron, nitrogen,oxygen, phosphorus, sulfur and selenium.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, andcycloalkyl substituted alkyl groups. In preferred embodiments, astraight chain or branched chain alkyl has 30 or fewer carbon atoms inits backbone (e.g., C₁-C₃₀ for straight chain, C₃-C₃₀ for branchedchain), and more preferably 20 or fewer. Likewise, preferred cycloalkylshave from 3-10 carbon atoms in their ring structure, and more preferablyhave 5, 6 or 7 carbons in the ring structure.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths. Preferred alkyl groups are lower alkyls. Inpreferred embodiments, a substituent designated herein as alkyl is alower alkyl.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group (e.g., an aromatic or heteroaromatic group).

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

The term “aryl” as used herein includes 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, anthracene, naphthalene, pyrene,pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole,pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.Those aryl groups having heteroatoms in the ring structure may also bereferred to as “aryl heterocycles” or “heteroaromatics.” The aromaticring can be substituted at one or more ring positions with suchsubstituents as described above, for example, halogen, azide, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester,heterocyclyl, aromatic or heteroaromatic moieties, —CF₃, —CN, or thelike. The term “aryl” also includes polycyclic ring systems having twoor more cyclic rings in which two or more carbons are common to twoadjoining rings (the rings are “fused rings”) wherein at least one ofthe rings is aromatic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.

The terms ortho, meta and para apply to 1,2-, 1,3- and 1,4-disubstitutedbenzenes, respectively. For example, the names 1,2-dimethylbenzene andortho-dimethylbenzene are synonymous.

The terms “heterocyclyl” or “heterocyclic group” refer to 3- to10-membered ring structures, more preferably 3- to 7-membered rings,whose ring structures include one to four heteroatoms. Heterocycles canalso be polycycles. Heterocyclyl groups include, for example, thiophene,thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, quinoline,phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine,phenanthroline, phenazine, phenarsazine, phenothiazine, furazan,phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine,piperazine, morpholine, lactones, lactams such as azetidinones andpyrrolidinones, sultams, sultones, and the like. The heterocyclic ringcan be substituted at one or more positions with such substituents asdescribed above, as for example, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic orheteroaromatic moiety, —CF₃, —CN, or the like.

The terms “polycyclyl” or “polycyclic group” refer to two or more rings(e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Rings that are joined throughnon-adjacent atoms are termed “bridged” rings. Each of the rings of thepolycycle can be substituted with such substituents as described above,as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromaticmoiety, —CF₃, —CN, or the like.

As used herein, the term “nitro” means —NO₂; the term “halogen”designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; the term“hydroxyl” means —OH; and the term “sulfonyl” means —SO₂—.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that can berepresented by the general formula:

wherein R₉, R₁₀ and R′₁₀ each independently represent a group permittedby the rules of valence.

The term “acylamino” is art-recognized and refers to a moiety that canbe represented by the general formula:

wherein R₉ is as defined above, and R′₁₁ represents a hydrogen, analkyl, an alkenyl or —(CH₂)_(m)—R₈, where m and R₈ are as defined above.

The term “amido” is art recognized as an amino-substituted carbonyl andincludes a moiety that can be represented by the general formula:

wherein R₉, R₁₀ are as defined above. Preferred embodiments of the amidewill not include imides which may be unstable.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In preferred embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl,—S-alkynyl, and —S—(CH₂)_(m)—R₈, wherein m and R₈ are defined above.Representative alkylthio groups include methylthio, ethyl thio, and thelike.

The term “carbonyl” is art recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R₁₁represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R₈ or apharmaceutically acceptable salt, R′₁₁ represents a hydrogen, an alkyl,an alkenyl or —(CH₂)_(m)—R₈, where m and R₈ are as defined above. WhereX is an oxygen and R₁₁ or R′₁₁ is not hydrogen, the formula representsan “ester”. Where X is an oxygen, and R₁₁ is as defined above, themoiety is referred to herein as a carboxyl group, and particularly whenR₁₁ is a hydrogen, the formula represents a “carboxylic acid”. Where Xis an oxygen, and R′₁₁ is hydrogen, the formula represents a “formate”.In general, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiolcarbonyl” group. Where X is asulfur and R₁₁ or R′₁₁ is not hydrogen, the formula represents a“thiolester.” Where X is a sulfur and R₁₁ is hydrogen, the formularepresents a “thiolcarboxylic acid.” Where X is a sulfur and R₁₁′ ishydrogen, the formula represents a “thiolformate.” On the other hand,where X is a bond, and R₁₁ is not hydrogen, the above formula representsa “ketone” group. Where X is a bond, and R₁₁ is hydrogen, the aboveformula represents an “aldehyde” group.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynyl, —O—(CH₂)_(m)—R₈,where m and R₈ are described above.

The term “sulfonate” is art recognized and includes a moiety that can berepresented by the general formula:

in which R₄₁ is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized andrefer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl,and nonafluorobutanesulfonyl groups, respectively. The terms triflate,tosylate, mesylate, and nonaflate are art-recognized and refer totrifluoromethanesulfonate ester, p-toluenesulfonate ester,methanesulfonate ester, and nonafluorobutanesulfonate ester functionalgroups and molecules that contain said groups, respectively.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl,phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations. The abbreviationscontained in said list, and all abbreviations utilized by organicchemists of ordinary skill in the art are hereby incorporated byreference.

The term “sulfate” is art recognized and includes a moiety that can berepresented by the general formula:

in which R₄₁ is as defined above.

The term “sulfonylamino” is art recognized and includes a moiety thatcan be represented by the general formula:

The term “sulfamoyl” is art-recognized and includes a moiety that can berepresented by the general formula:

The term “sulfonyl”, as used herein, refers to a moiety that can berepresented by the general formula:

in which R₄₄ is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl.

The term “sulfoxido” as used herein, refers to a moiety that can berepresented by the general formula:

in which R₄₄ is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl.

A “selenoalkyl” refers to an alkyl group having a substituted selenogroup attached thereto. Exemplary “selenoethers” which may besubstituted on the alkyl are selected from one of —Se-alkyl,—Se-alkenyl, —Se-alkynyl, and —Se—(CH₂)_(m)—R₇, m and R₇ being definedabove.

Analogous substitutions can be made to alkenyl and alkynyl groups toproduce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

As used herein, the definition of each expression, e.g. alkyl, m, n,etc., when it occurs more than once in any structure, is intended to beindependent of its definition elsewhere in the same structure.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described herein above. The permissible substituentscan be one or more and the same or different for appropriate organiccompounds. For purposes of this invention, the heteroatoms such asnitrogen may have hydrogen substituents and/or any permissiblesubstituents of organic compounds described herein which satisfy thevalences of the heteroatoms. This invention is not intended to belimited in any manner by the permissible substituents of organiccompounds.

The phrase “protecting group” as used herein means temporarysubstituents which protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed (Greene, T. W.; Wuts,P.G.M. Protective Groups in Organic Synthesis, 2^(nd) ed.; Wiley: NewYork, 1991).

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

Contemplated equivalents of the compounds described above includecompounds which otherwise correspond thereto, and which have the samegeneral properties thereof (e.g., functioning as analgesics), whereinone or more simple variations of substituents are made which do notadversely affect the efficacy of the compound in binding to sigmareceptors. In general, the compounds of the present invention may beprepared by the methods illustrated in the general reaction schemes as,for example, described below, or by modifications thereof, using readilyavailable starting materials, reagents and conventional synthesisprocedures. In these reactions, it is also possible to make use ofvariants which are in themselves known, but are not mentioned here.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.

Pharmaceutical Compositions

In another aspect, the present invention provides pharmaceuticallyacceptable compositions which comprise a therapeutically-effectiveamount of one or more of the compounds described above, formulatedtogether with one or more pharmaceutically acceptable carriers(additives) and/or diluents. As described in detail below, thepharmaceutical compositions of the present invention may be speciallyformulated for administration in solid or liquid form, including thoseadapted for the following: (1) oral administration, for example,drenches (aqueous or non-aqueous solutions or suspensions), tablets,e.g., those targeted for buccal, sublingual, and systemic absorption,boluses, powders, granules, pastes for application to the tongue; (2)parenteral administration, for example, by subcutaneous, intramuscular,intravenous or epidural injection as, for example, a sterile solution orsuspension, or sustained-release formulation; (3) topical application,for example, as a cream, ointment, or a controlled-release patch orspray applied to the skin; (4) intravaginally or intrarectally, forexample, as a pessary, cream or foam; (5) sublingually; (6) ocularly;(7) transdermally; or (8) nasally.

The phrase “therapeutically-effective amount” as used herein means thatamount of a compound, material, or composition comprising a compound ofthe present invention which is effective for producing some desiredtherapeutic effect in at least a sub-population of cells in an animal ata reasonable benefit/risk ratio applicable to any medical treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient. Some examples of materials which canserve as pharmaceutically-acceptable carriers include: (1) sugars, suchas lactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; and (22) othernon-toxic compatible substances employed in pharmaceutical formulations.

As set out above, certain embodiments of the present compounds maycontain a basic functional group, such as amino or alkylamino, and are,thus, capable of forming pharmaceutically-acceptable salts withpharmaceutically-acceptable acids. The term “pharmaceutically-acceptablesalts” in this respect, refers to the relatively non-toxic, inorganicand organic acid addition salts of compounds of the present invention.These salts can be prepared in situ in the administration vehicle or thedosage form manufacturing process, or by separately reacting a purifiedcompound of the invention in its free base form with a suitable organicor inorganic acid, and isolating the salt thus formed during subsequentpurification. Representative salts include the hydrobromide,hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate,valerate, oleate, palmitate, stearate, laurate, benzoate, lactate,phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate,napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonatesalts and the like. (See, for example, Berge et al. (1977)“Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19)

The pharmaceutically acceptable salts of the subject compounds includethe conventional nontoxic salts or quaternary ammonium salts of thecompounds, e.g., from non-toxic organic or inorganic acids. For example,such conventional nontoxic salts include those derived from inorganicacids such as hydrochloride, hydrobromic, sulfuric, sulfamic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically-acceptable salts with pharmaceutically-acceptablebases. The term “pharmaceutically-acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of compounds of the present invention. These salts can likewise beprepared in situ in the administration vehicle or the dosage formmanufacturing process, or by separately reacting the purified compoundin its free acid form with a suitable base, such as the hydroxide,carbonate or bicarbonate of a pharmaceutically-acceptable metal cation,with ammonia, or with a pharmaceutically-acceptable organic primary,secondary or tertiary amine. Representative alkali or alkaline earthsalts include the lithium, sodium, potassium, calcium, magnesium, andaluminum salts and the like. Representative organic amines useful forthe formation of base addition salts include ethylamine, diethylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.(See, for example, Berge et al., supra)

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated, the particular mode ofadministration. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect.Generally, out of one hundred percent, this amount will range from about0.1 percent to about ninety-nine percent of active ingredient,preferably from about 5 percent to about 70 percent, most preferablyfrom about 10 percent to about 30 percent.

In certain embodiments, a formulation of the present invention comprisesan excipient selected from the group consisting of cyclodextrins,celluloses, liposomes, micelle forming agents, e.g., bile acids, andpolymeric carriers, e.g., polyesters and polyanhydrides; and a compoundof the present invention. In certain embodiments, an aforementionedformulation renders orally bioavailable a compound of the presentinvention.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules, trouches and thelike), the active ingredient is mixed with one or morepharmaceutically-acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds and surfactants, such as poloxamer and sodium laurylsulfate; (7) wetting agents, such as, for example, cetyl alcohol,glycerol monostearate, and non-ionic surfactants; (8) absorbents, suchas kaolin and bentonite clay; (9) lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, zinc stearate, sodium stearate, stearic acid, and mixturesthereof; (10) coloring agents; and (11) controlled release agents suchas crospovidone or ethyl cellulose. In the case of capsules, tablets andpills, the pharmaceutical compositions may also comprise bufferingagents. Solid compositions of a similar type may also be employed asfillers in soft and hard-shelled gelatin capsules using such excipientsas lactose or milk sugars, as well as high molecular weight polyethyleneglycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be formulated for rapid release,e.g., freeze-dried. They may be sterilized by, for example, filtrationthrough a bacteria-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedin sterile water, or some other sterile injectable medium immediatelybefore use. These compositions may also optionally contain opacifyingagents and may be of a composition that they release the activeingredient(s) only, or preferentially, in a certain portion of thegastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically-acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane or dispersing the compoundin a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containsugars, alcohols, antioxidants, buffers, bacteriostats, solutes whichrender the formulation isotonic with the blood of the intended recipientor suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms upon the subject compounds may be ensuredby the inclusion of various antibacterial and antifungal agents, forexample, paraben, chlorobutanol, phenol sorbic acid, and the like. Itmay also be desirable to include isotonic agents, such as sugars, sodiumchloride, and the like into the compositions. In addition, prolongedabsorption of the injectable pharmaceutical form may be brought about bythe inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99% (morepreferably, 10 to 30%) of active ingredient in combination with apharmaceutically acceptable carrier.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given in formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, etc. administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Oral administrations are preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically-acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion ormetabolism of the particular compound being employed, the rate andextent of absorption, the duration of the treatment, other drugs,compounds and/or materials used in combination with the particularcompound employed, the age, sex, weight, condition, general health andprior medical history of the patient being treated, and like factorswell known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. Generally, oral, intravenous,intracerebroventricular and subcutaneous doses of the compounds of thisinvention for a patient, when used for the indicated analgesic effects,will range from about 0.0001 to about 100 mg per kilogram of body weightper day.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms. Preferred dosing is one administrationper day.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical formulation (composition).

The compounds according to the invention may be formulated foradministration in any convenient way for use in human or veterinarymedicine, by analogy with other pharmaceuticals.

In another aspect, the present invention provides pharmaceuticallyacceptable compositions which comprise a therapeutically-effectiveamount of one or more of the subject compounds, as described above,formulated together with one or more pharmaceutically acceptablecarriers (additives) and/or diluents. As described in detail below, thepharmaceutical compositions of the present invention may be speciallyformulated for administration in solid or liquid form, including thoseadapted for the following: (1) oral administration, for example,drenches (aqueous or non-aqueous solutions or suspensions), tablets,boluses, powders, granules, pastes for application to the tongue; (2)parenteral administration, for example, by subcutaneous, intramuscularor intravenous injection as, for example, a sterile solution orsuspension; (3) topical application, for example, as a cream, ointmentor spray applied to the skin, lungs, or mucous membranes; or (4)intravaginally or intrarectally, for example, as a pessary, cream orfoam; (5) sublingually or buccally; (6) ocularly; (7) transdermally; or(8) nasally.

The term “treatment” is intended to encompass also prophylaxis, therapyand cure.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; and poultry and pets in general.

The compound of the invention can be administered as such or inadmixtures with pharmaceutically acceptable carriers and can also beadministered in conjunction with antimicrobial agents such aspenicillins, cephalosporins, aminoglycosides and glycopeptides.Conjunctive therapy, thus includes sequential, simultaneous and separateadministration of the active compound in a way that the therapeuticaleffects of the first administered one is not entirely disappeared whenthe subsequent is administered.

The addition of the active compound of the invention to animal feed ispreferably accomplished by preparing an appropriate feed premixcontaining the active compound in an effective amount and incorporatingthe premix into the complete ration.

Alternatively, an intermediate concentrate or feed supplement containingthe active ingredient can be blended into the feed. The way in whichsuch feed premixes and complete rations can be prepared and administeredare described in reference books (such as “Applied Animal Nutrition”,W.H. Freedman and CO., San Francisco, U.S.A., 1969 or “Livestock Feedsand Feeding” O and B books, Corvallis, Ore., U.S.A., 1977).

Micelles

Recently, the pharmaceutical industry introduced microemulsificationtechnology to improve bioavailability of some lipophilic (waterinsoluble) pharmaceutical agents. Examples include Trimetrine (Dordunoo,S. K., et al., Drug Development and Industrial Pharmacy, 17(12),1685-1713, 1991 and REV 5901 (Sheen, P. C., et al., J Pharm Sci 80(7),712-714, 1991). Among other things, microemulsification providesenhanced bioavailability by preferentially directing absorption to thelymphatic system instead of the circulatory system, which therebybypasses the liver, and prevents destruction of the compounds in thehepatobiliary circulation.

In one aspect of invention, the formulations contain micelles formedfrom a compound of the present invention and at least one amphiphiliccarrier, in which the micelles have an average diameter of less thanabout 100 nm. More preferred embodiments provide micelles having anaverage diameter less than about 50 nm, and even more preferredembodiments provide micelles having an average diameter less than about30 nm, or even less than about 20 nm.

While all suitable amphiphilic carriers are contemplated, the presentlypreferred carriers are generally those that haveGenerally-Recognized-as-Safe (GRAS) status, and that can both solubilizethe compound of the present invention and microemulsify it at a laterstage when the solution comes into a contact with a complex water phase(such as one found in human gastro-intestinal tract). Usually,amphiphilic ingredients that satisfy these requirements have HLB(hydrophilic to lipophilic balance) values of 2-20, and their structurescontain straight chain aliphatic radicals in the range of C-6 to C-20.Examples are polyethylene-glycolized fatty glycerides and polyethyleneglycols.

Particularly preferred amphiphilic carriers are saturated andmonounsaturated polyethyleneglycolyzed fatty acid glycerides, such asthose obtained from fully or partially hydrogenated various vegetableoils. Such oils may advantageously consist of tri-. di- and mono-fattyacid glycerides and di- and mono-polyethyleneglycol esters of thecorresponding fatty acids, with a particularly preferred fatty acidcomposition including capric acid 4-10, capric acid 3-9, lauric acid40-50, myristic acid 14-24, palmitic acid 4-14 and stearic acid 5-15%.Another useful class of amphiphilic carriers includes partiallyesterified sorbitan and/or sorbitol, with saturated or mono-unsaturatedfatty acids (SPAN-series) or corresponding ethoxylated analogs(TWEEN-series).

Commercially available amphiphilic carriers are particularlycontemplated, including Gelucire-series, Labrafil, Labrasol, orLauroglycol (all manufactured and distributed by Gattefosse Corporation,Saint Priest, France), PEG-mono-oleate, PEG-di-oleate, PEG-mono-laurateand di-laurate, Lecithin, Polysorbate 80, etc (produced and distributedby a number of companies in USA and worldwide).

Polymers

Hydrophilic polymers suitable for use in the present invention are thosewhich are readily water-soluble, can be covalently attached to avesicle-forming lipid, and which are tolerated in vivo without toxiceffects (i.e., are biocompatible). Suitable polymers includepolyethylene glycol (PEG), polylactic (also termed polylactide),polyglycolic acid (also termed polyglycolide), a polylactic-polyglycolicacid copolymer, and polyvinyl alcohol. Preferred polymers are thosehaving a molecular weight of from about 100 or 120 daltons up to about5,000 or 10,000 daltons, and more preferably from about 300 daltons toabout 5,000 daltons. In a particularly preferred embodiment, the polymeris polyethyleneglycol having a molecular weight of from about 100 toabout 5,000 daltons, and more preferably having a molecular weight offrom about 300 to about 5,000 daltons. In a particularly preferredembodiment, the polymer is polyethyleneglycol of 750 daltons (PEG(750)).Polymers may also be defined by the number of monomers therein; apreferred embodiment of the present invention utilizes polymers of atleast about three monomers, such PEG polymers consisting of threemonomers (approximately 150 daltons).

Other hydrophilic polymers which may be suitable for use in the presentinvention include polyvinylpyrrolidone, polymethoxazoline,polyethyloxazoline, polyhydroxypropyl methacrylamide,polymethacrylamide, polydimethylacrylamide, and derivatized cellulosessuch as hydroxymethylcellulose or hydroxyethylcellulose.

In certain embodiments, a formulation of the present invention comprisesa biocompatible polymer selected from the group consisting ofpolyamides, polycarbonates, polyalkylenes, polymers of acrylic andmethacrylic esters, polyvinyl polymers, polyglycolides, polysiloxanes,polyurethanes and co-polymers thereof, celluloses, polypropylene,polyethylenes, polystyrene, polymers of lactic acid and glycolic acid,polyanhydrides, poly(ortho)esters, poly(butic acid), poly(valeric acid),poly(lactide-co-caprolactone), polysaccharides, proteins, polyhyaluronicacids, polycyanoacrylates, and blends, mixtures, or copolymers thereof.

Cyclodextrins

Cyclodextrins are cyclic oligosaccharides, consisting of 6, 7 or 8glucose units, designated as alpha, beta or gamma, respectively.Cyclodextrins with fewer than six glucose units are not known to exist.The glucose units are linked by alpha-1,4-glucosidic bonds. As aconsequence of the chair conformation of the sugar units, all secondaryhydroxyl groups (at C-2, C-3) are located on one side of the ring, whileall the primary hydroxyl groups at C-6 are situated on the other side.As a result, the external faces are hydrophilic, making thecyclodextrins water-soluble. In contrast, the cavities of thecyclodextrins are hydrophobic, since they are lined by the hydrogen ofatoms C-3 and C-5, and by ether-like oxygens. These matrices allowcomplexation with a variety of relatively hydrophobic compounds,including, for instance, steroid compounds such as 17-beta-estradiol(see, e.g., van Uden et al. Plant Cell Tiss. Org. Cult. 38:1-3-113(1994)). The complexation takes place by Van der Waals interactions andby hydrogen bond formation. For a general review of the chemistry ofcyclodextrins, see, Wenz, Agnew. Chem. Int. Ed. Engl., 33:803-822(1994).

The physico-chemical properties of the cyclodextrin derivatives dependstrongly on the kind and the degree of substitution. For example, theirsolubility in water ranges from insoluble (e.g.,triacetyl-beta-cyclodextrin) to 147% soluble (w/v)(G-2-beta-cyclodextrin). In addition, they are soluble in many organicsolvents. The properties of the cyclodextrins enable the control oversolubility of various formulation components by increasing or decreasingtheir solubility.

Numerous cyclodextrins and methods for their preparation have beendescribed. For example, Parmeter (I), et al. (U.S. Pat. No. 3,453,259)and Gramera, et al. (U.S. Pat. No. 3,459,731) described electroneutralcyclodextrins. Other derivatives include cyclodextrins with cationicproperties [Parmeter (II), U.S. Pat. No. 3,453,257], insolublecrosslinked cyclodextrins (Sohms, U.S. Pat. No. 3,420,788), andcyclodextrins with anionic properties [Parmeter (III), U.S. Pat. No.3,426,011]. Among the cyclodextrin derivatives with anionic properties,carboxylic acids, phosphorous acids, phosphinous acids, phosphonicacids, phosphoric acids, thiophosphonic acids, thiosulphinic acids, andsulfonic acids have been appended to the parent cyclodextrin [see,Parmeter (III), supra]. Furthermore, sulfoalkyl ether cyclodextrinderivatives have been described by Stella, et al. (U.S. Pat. No.5,134,127).

Liposomes

Liposomes consist of at least one lipid bilayer membrane enclosing anaqueous internal compartment. Liposomes may be characterized by membranetype and by size. Small unilamellar vesicles (SUVs) have a singlemembrane and typically range between 0.02 and 0.05 μM in diameter; largeunilamellar vesicles (LUVS) are typically larger than 0.05 μmOligolamellar large vesicles and multilamellar vesicles have multiple,usually concentric, membrane layers and are typically larger than 0.1μm. Liposomes with several nonconcentric membranes, i.e., severalsmaller vesicles contained within a larger vesicle, are termedmultivesicular vesicles.

One aspect of the present invention relates to formulations comprisingliposomes containing a compound of the present invention, where theliposome membrane is formulated to provide a liposome with increasedcarrying capacity. Alternatively or in addition, the compound of thepresent invention may be contained within, or adsorbed onto, theliposome bilayer of the liposome. The compound of the present inventionmay be aggregated with a lipid surfactant and carried within theliposome's internal space; in these cases, the liposome membrane isformulated to resist the disruptive effects of the activeagent-surfactant aggregate.

According to one embodiment of the present invention, the lipid bilayerof a liposome contains lipids derivatized with polyethylene glycol(PEG), such that the PEG chains extend from the inner surface of thelipid bilayer into the interior space encapsulated by the liposome, andextend from the exterior of the lipid bilayer into the surroundingenvironment.

Active agents contained within liposomes of the present invention are insolubilized form. Aggregates of surfactant and active agent (such asemulsions or micelles containing the active agent of interest) may beentrapped within the interior space of liposomes according to thepresent invention. A surfactant acts to disperse and solubilize theactive agent, and may be selected from any suitable aliphatic,cycloaliphatic or aromatic surfactant, including but not limited tobiocompatible lysophosphatidylcholines (LPCs) of varying chain lengths(for example, from about C14 to about C20). Polymer-derivatized lipidssuch as PEG-lipids may also be utilized for micelle formation as theywill act to inhibit micelle/membrane fusion, and as the addition of apolymer to surfactant molecules decreases the CMC of the surfactant andaids in micelle formation. Preferred are surfactants with CMCs in themicromolar range; higher CMC surfactants may be utilized to preparemicelles entrapped within liposomes of the present invention, however,micelle surfactant monomers could affect liposome bilayer stability andwould be a factor in designing a liposome of a desired stability.

Liposomes according to the present invention may be prepared by any of avariety of techniques that are known in the art. See, e.g., U.S. Pat.No. 4,235,871; Published PCT application WO 96/14057; New RRC,Liposomes: A practical approach, IRL Press, Oxford (1990), pages 33-104;Lasic DD, Liposomes from physics to applications, Elsevier SciencePublishers BV, Amsterdam, 1993.

For example, liposomes of the present invention may be prepared bydiffusing a lipid derivatized with a hydrophilic polymer into preformedliposomes, such as by exposing preformed liposomes to micelles composedof lipid-grafted polymers, at lipid concentrations corresponding to thefinal mole percent of derivatized lipid which is desired in theliposome. Liposomes containing a hydrophilic polymer can also be formedby homogenization, lipid-field hydration, or extrusion techniques, asare known in the art.

In another exemplary formulation procedure, the active agent is firstdispersed by sonication in a lysophosphatidylcholine or other low CMCsurfactant (including polymer grafted lipids) that readily solubilizeshydrophobic molecules. The resulting micellar suspension of active agentis then used to rehydrate a dried lipid sample that contains a suitablemole percent of polymer-grafted lipid, or cholesterol. The lipid andactive agent suspension is then formed into liposomes using extrusiontechniques as are known in the art, and the resulting liposomesseparated from the unencapsulated solution by standard columnseparation.

In one aspect of the present invention, the liposomes are prepared tohave substantially homogeneous sizes in a selected size range. Oneeffective sizing method involves extruding an aqueous suspension of theliposomes through a series of polycarbonate membranes having a selecteduniform pore size; the pore size of the membrane will correspond roughlywith the largest sizes of liposomes produced by extrusion through thatmembrane. See, e.g., U.S. Pat. No. 4,737,323.

Release Modifiers

The release characteristics of a formulation of the present inventiondepend on the encapsulating material, the concentration of encapsulateddrug, and the presence of release modifiers. For example, release can bemanipulated to be pH dependent, for example, using a pH sensitivecoating that releases only at a low pH, as in the stomach, or a higherpH, as in the intestine. An enteric coating can be used to preventrelease from occurring until after passage through the stomach. Multiplecoatings or mixtures of cyanamide encapsulated in different materialscan be used to obtain an initial release in the stomach, followed bylater release in the intestine. Release can also be manipulated byinclusion of salts or pore forming agents, which can increase wateruptake or release of drug by diffusion from the capsule. Excipientswhich modify the solubility of the drug can also be used to control therelease rate. Agents which enhance degradation of the matrix or releasefrom the matrix can also be incorporated. They can be added to the drug,added as a separate phase (i.e., as particulates), or can beco-dissolved in the polymer phase depending on the compound. In allcases the amount should be between 0.1 and thirty percent (w/w polymer).Types of degradation enhancers include inorganic salts such as ammoniumsulfate and ammonium chloride, organic acids such as citric acid,benzoic acid, and ascorbic acid, inorganic bases such as sodiumcarbonate, potassium carbonate, calcium carbonate, zinc carbonate, andzinc hydroxide, and organic bases such as protamine sulfate, spermine,choline, ethanolamine, diethanolamine, and triethanolamine andsurfactants, such as Tween® and Pluronic®. Pore forming agents which addmicrostructure to the matrices (i.e., water soluble compounds such asinorganic salts and sugars) are added as particulates. The range shouldbe between one and thirty percent (w/w polymer).

Uptake can also be manipulated by altering residence time of theparticles in the gut. This can be achieved, for example, by coating theparticle with, or selecting as the encapsulating material, a mucosaladhesive polymer. Examples include most polymers with free carboxylgroups, such as chitosan, celluloses, and especially polyacrylates (asused herein, polyacrylates refers to polymers including acrylate groupsand modified acrylate groups such as cyanoacrylates and methacrylates).

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1

Monomer Synthesis

^(a) (i) X—P(Cl)—N(iPr)₂, TEA/dichloromethane or X—P—[N(iPr)₂]₂,tetrazole/tetrazole-diisopropylammonium salt/MeCN; or X—P(Cl₂), one eq.HN(iPr)₂ followed by one 1 eq. 1 in MeCN/dichloromethane.Synthesis of Methylphosphonamidite [2, R=OTBDMS, X=Me, B=Adenine (N-bz);Cytosine (N—Ac); 5-Methylcytosine (N-Bz); Uracil; 5-Methyluracil;Guanine (N-iBu); or Inosine, Scheme 1):Method 1

Appropriately base protected 5′-O-DMT-2′-O-TBDS-nucleoside (adenosine,guanosine, cytidine, 5-methylcytidine, uridine, 5-methyluridine orinosine) purchased from ChemGenes Corporation, 33 Industrial Way,Wilmington, Mass. is reacted with 1.2 equivalent ofchloro-N,N-diisopropylaminomethylphosphine (obtained from from ChemGenesCorporation, 33 Industrial Way, Wilmington, Mass.) in anhydrousdichloromethane (or THF) containing 3.0 equivalent ofdiisopropylethylamine to obtain a diastereomeric mixture of the desiredmethylphosphonamidite 2 as reported by Sinha et al. (Nucleic Acids Res.,1994, 22, 3119).

(N-Bz)-5′-O-DMT-2′-O-TBDMS-Adenosine-3′-O—(P-Methyl)phosphonamidite: Toa solution of (N-Bz)-5′-O-DMT-2′-O-TBDMS-adenosine (5 g, 6.4 mmol) inanhydrous dichloromethane (50 mL) were added anhydrous diisopropyl amine(2.06 g, 2.8 mL, 16 mmol) andmethyl-N,N′-diisopropylamino-chlorophosphine (2.3 g, 2.3 mL, 12.8 mmol)under argon atm. The reaction mixture was stirred at rt for 16 h. It wasthen diluted with dichloromethane (50 mL) and poured into ice-cold water(50 mL), shaken and separated. The aqueous layer was extracted withdichloromethane (3×25 mL). The combined organic layer was dried overanhydrous sodium sulfate and filtered. Upon removal of solvent underreduced pressure, 5.5 g of crude product was obtained. The crude productwas subjected to flash column chromatography over silica gel.Hexane:EtOAc:Et₃N (74:25:1) mixture was used to elute the product. Theproduct was obtained as a white foamy solid. (4.4 g, 74%). ¹H NMR (400MHz, CDCl₃): δ 8.68 (s, 1H), 7.98 (s, 1H), 7.7.-7.8 (m, 4H), 7.54 (m,4H), 7.2 (m, 2H), 7.05 (m, 2H), 6.94 (m, 2H), 6.78 (m, 4H), 6.2 (d, 1H),6.08 (d, 1H, minor diastereomer), 5.45 (m, 1H), 5.16 (m, 1H, minordiastereomer), 4.58 (m, 2H), 3.78 (m, 1H), 3.64 (m, 1H), 3.32 (s, 6H),2.4 (m, 2H), 1.4 (d, 2H), 1.05 (m, 7H), 0.9-1.0 (m, 8H), 0.8 (s, 9H),0.2 (s, 3H). ³¹P NMR (161.82 MHz, CDCl₃): δ 122.01, 118.81. ¹³C NMR (100MHz, CDCl₃): δ 159.23, 159.21, 152.18, 150.82, 145.55, 142.44, 136.31,136.26, 136.14, 136.07, 134.79, 131.9, 130.67, 130.64, 128.85, 128.65,128.12, 127.88, 127.19, 127.12, 124.58, 113.61, 88.96, 87.25, 85.28,76.88, 76.73, 75.3, 64.35, 60.02, 54.78, 46.71, 44.43, 44.33, 25.93,25.87, 20.52, 18.3, 18.15, 17.96, 17.84, 17.72, 14.17, 12.35.

(N-iBU)-5′-O-DMT-2′-O-TBDMS-Guanosine-3′-O-(E-Methyl)phosphonamidite: Toa solution of (N-iBu)-5′-O-DMT-2′-O-TBDMS-guanosine (10 g, 12.98 mmol)in anhydrous dichloromethane (75 mL) were added anhydrous diisopropylamine (5.03 g, 6.78 mL, 38.94 mmol) andmethyl-N,N′-diisopropylamino-chlorophosphine (4.7 g, 4.7 mL, 25.9 mmol)under argon atm. The reaction mixture was stirred at rt for 5 h. It wasthen diluted with dichloromethane (100 mL) and poured into ice-coldwater (50 mL), shaken and separated. The aqueous layer was extractedwith dichloromethane (3×50 mL). The combined organic layer was driedover anhydrous sodium sulfate and filtered. Upon removal of solventunder reduced pressure, 11.1 g of crude product was obtained. The crudeproduct was subjected to flash column chromatography over silica gel.Hexane:EtOAc:Et₃N (59:40:1) mixture was used to elute the product. Theproduct was obtained as a white foamy solid. (9.0 g, 73%). ¹H NMR (400MHz, CDCl₃): δ 7.84 (s, 1H), 7.72 (d, 2H), 7.56 (m, 5H), 7.0-7.2 (m,4H), 6.8 (m, 5H), 6.16 (d, 1H, minor diastereomer), 6.02 (d, 1H), 5.38(m, 1H), 5.02 (m, 1H, minor diastereomer), 4.72 (m, 1H, minordiastereomer), 4.58 (m, 1H, minor diastereomer), 3.98 (s, 11H), 3.88 (m,1H, minor diastereomer), 3.72 (m, 1H), 3.32 (d, 6H), 2.3 (s, 1H), 2.1(s, 1H), 1.5 (m, 1H), 1.36 (d, 2H), 1.0 (m, 9H), 0.9 (s, 9H). ³¹P NMR(161.82 MHz, CDCl₃): δ 122.53, 115.88.

(N—Ac)-5′-O-DMT-2′-O-TBDMS-Cytidine-3′-O—(P-Methyl)phosphonamidite: Thedesired phosphonamidite (4.80 g, 66.3%) was prepared from(N—Ac)-5′-O-DMT-2′-O-TBDMS-Cytidine (6.0 g, 8.548 mmol) andmethyl-N,N′-diisopropylamino-chlorophosphine (5.0 g, 27.526 mmol) in thepresence of diisopropylethylamine (9 mL, 51.66 mmol) and purified asdescribed above. ³¹P NMR (161.81 MHz, CDCl₃): δ 122.54, 121.63

5-Me-5′-O-DMT-2′-O-TBDMS-Uridine-3′-O—(P-Methyl)phosphonamidite: Thedesired phosphonamidite (1.90 g, 52.1%) was prepared from5-Me-5′-O-DMT-2′-O-TBDMS-Uridine (3.0 g, 4.449 mmol) andmethyl-N,N′-diisopropylamino-chlorophosphine (1.7 g, 9.354 mmol) in thepresence of diisopropylethylamine (2.4 mL, 13.777 mmol) and purified asdescribed above. ³¹P NMR (161.81 MHz, CDCl₃): δ 124.12, 117.38

Method 2

Appropriately base protected 5′-O-DMT-2′-O-TBDS-nucleoside (adenosine,guanosine, cytidine, 5-methylcytidine, uridine, 5-methyluridine orinosine) purchased from ChemGenes Corporation, 33 Industrial Way,Wilmington, Mass. is reacted with bis(diisopropylamino)methylphosphineand 1H-tetrazole in acetonitrile to obtain a diastereomeric mixture ofthe desired methylphosphonamidite 2 as reported in the literature(Lauritsen et al., Bioorg. Med. Chem. Lett., 2003, 13, 253). Thebis(diisopropylamino)methylphosphine reagent is obtained as reportedMoriarty et al. (J. Am. Chem. Soc., 1990, 112, 8575).

Method 3

Methyldichlorophosphine (purchased from Aldrich) is reacted with oneequivalent of anhydrous diisopropylamine (purchased from Aldrich) indichloromethane containing three equivalent of diisopropylethylamine. Tothe resulting reaction mixture one equivalent of the appropriatelyprotected nucleoside is added under constant stirring at ambienttemperature. The diastereomeric mixture of the desiredmethylphosphonamidite after standard workup is purified by flash silicagel column chromatography to obtain pure compound 2 (Vaghefi et al.,Nucleic Acids Res., 1995, 23, 3600).

Example 2

Synthesis of Isopropylphosphonamidite [2, R=H, OTBDMS or OMe,X=Isopropyl, B=Adenine (N-bz); Cytosine (N—Ac); 5-Methylcytosine (N-Bz);Uracil; 5-Methyluracil; Guanine (N-iBu); or Inosine, Scheme 1):

Isopropyldichlorophosphine (purchased from Aldrich) is reacted with oneequivalent of anhydrous diisopropylamine (purchased from Aldrich) indichloromethane containing three equivalent of diisopropylethylamine. Tothe resulting reaction mixture one equivalent of the appropriatelyprotected nucleoside is added under constant stirring at ambienttemperature. The diastereomeric mixture of the desiredisopropylphosphonamidite after standard workup is purified by flashsilica gel column chromatography to obtain pure compound 2 (Vaghefi etal., Nucleic Acids Res., 1995, 23, 3600).

5′-O-DMT-2′-deoxy-thymidine-3′-O—(P-isopropyl)phosohonamidite: To asolution of i-Pr₂NH (0.31 ml) in dry CH₃CN (4 mL) at −20° C. was addeddichloroisopropylphosphine (11 uL) and stirred at the same temperaturefor 20 to 40 min. 5′-DMTr-2′-deoxy-T (500 mg) in dry dichloromethane(2-3 mL) and triethylamine (0.18 mL) were added and stirred at RT for 16h under an argon atmosphere. The reaction mixture was concentrated intoa crude which was applied to a column of silica gel eluted withhexanes-ethyl acetate (1:1) to give a pure compound (450 mg, 65%) as twoisomers. ³¹P-NMR (CDCl₃, 400 MHz): δ 133.61, 132.17.

Example 3

Synthesis of tert-Butylphosphonamidite [2, R=H, OTBDMS or OMe,X=tert-Butyl, B=Adenine (N-bz); Cytosine (N—Ac); 5-Methylcytosine(N-Bz); Uracil; 5-Methyluracil; Guanine (N-iBu); or Inosine, Scheme 1):

tert-Butyldichlorophosphine (purchased from Aldrich) is reacted with oneequivalent of anhydrous diisopropylamine (purchased from Aldrich) indichloromethane containing three equivalent of diisopropylethylamine. Tothe resulting reaction mixture one equivalent of the appropriatelyprotected nucleoside is added under constant stirring at ambienttemperature. The diastereomeric mixture of the desiredtert-butylphosphonamidite after standard workup is purified by flashsilicagel column chromatography to obtain pure compound 2 (Vaghefi etal., Nucleic Acids Res., 1995, 23, 3600).

5′-O-DMT-2′-deoxy-thymidine-3′-O-(E-tert-butyl)phosphonamidite: To asolution of i-Pr₂NH (6.17 ml) in dry CH₃CN (50 mL) at −20° C. was addedtert-butyldichlorophosphine (3.0 g) and stirred at −20 to 0° C. for 20to 40 min. 5′-DMTr-2′-deoxy-T (10 g) in dry dichloromethane (50 mL) andtriethylamine (3.56 mL) were added and stirred at RT for 4-6 days underan argon atmosphere. The reaction mixture was concentrated into a crudewhich was applied to a column of silica gel eluted with hexanes-ethylacetate (1:1) to give a pure compound (200 mg, 1.42%) as two isomers.One isomer NMR data: ¹H-NMR (CDCl₃, 400 MHz): δ 8.15 (br, NH), 7.58 (s,1H), 7.40-7.20 (m, 9H, ArH),6.80 (d, 4H), 6.50-6.40 (dd, 1H, H′-1), 5.40(t, 1H), 4.23 (dd, 1H), 3.79 (s, 6H, 2 OCH3), 3.50-3.38 (m, 2H, H′-5a,H′-5b), 2.80 (dd, 1H, H′-2a), 2.50-2.40 (m, 1H, H′-2b), 2.10 (s, 3H,5-CH3), 1.50-1.00 (m, 23H). ³¹P-NMR (CDCl₃, 400 MHz): δ 133.47.

Example 4

Synthesis of Methylphosphonamidite of Psuedouridine (2, R=H or OTBDMSand Z=H, Scheme 1):

5′-O-DMT-2′-O-TBDMS pseudouridine is obtained as reported in theliterature (Hall and McLaughlin, Nucleic Acids Res., 1992, 20, 1883).The desired psuedouridine methylphonamidite is prepared from5′-O-DMT-2′-O-TBDMS pseudouridine andchloro-N,N-diisopropylaminomethylphosphine as described in Example 1,method 2.

Synthesis of Isopropylphosphonamidite of Psuedouridine (2, R=H or OTBDMSand Z=H, Scheme 1):

5′-O-DMT-2′-O-TBDMS pseudouridine is obtained as reported in theliterature (Hall and McLaughlin, Nucleic Acids Res., 1992, 20, 1883).The desired psuedouridine isopropylphonamidite 2 is prepared from5′-O-DMT-2′-O-TBDMS pseudouridine and dichloroisopropylphosphine asdescribed in Example 1, method 3.

Synthesis of Methylphosphonamidite of Psuedouridine (2, R=OMe and Z=H,Scheme 1):

5′-O-DMT-2′-O-Me pseudouridine is obtained as reported in the literature(Ross et al., Nucleosides Nucleotides, 1997, 16, 1547). The desiredpsuedouridine methylphonamidite 2 is prepared from 5′-O-DMT-2′-O-Mepseudouridine and chloro-N,N-diisopropylaminomethylphosphine asdescribed in Example 1, method 2.

Synthesis of Isopropylphosphonamidite of Psuedouridine (2, R=OMe andZ=H, Scheme 1):

5′-O-DMT-2′-O-Me pseudouridine is obtained as reported in the literature(Ross et al., Nucleosides Nucleotides, 1997, 16, 1547). The desiredpsuedouridine isopropylphonamidite 2 is prepared from 5′-O-DMT-2′-O-Mepseudouridine and dichloroisopropylphosphine as described in Example 1,method 3.

Example 5

Synthesis of Methylphosphonamidite of 2′-O-Me-2-thiouridine (2, Scheme1):

5′-O-DMT-2′-O-Me-2-thiouridine is obtained as reported in the literature(Shoda et al., Bioorg. Med. Chem. Lett., 2000, 10, 1795). The desiredmethylphoanmidite 2 is is prepared from 5′-O-DMT-2′-O-Me-2-thiouridineand chloro-N,N-diisopropylaminomethylphosphine as described in Example1, method 2.

Synthesis of Isopropylphosphonamidite of 2′-O-Me-2-thiouridine (2,Scheme 1):

5′-O-DMT-2′-O-Me-2-thiouridine is obtained as reported in the literature(Shoda et al., Bioorg. Med. Chem. Lett., 2000, 10, 1795). The desiredisopropylphoanmidite 2 is prepared from 5′-O-DMT-2′-O-Me-2-thiouridineand dichloroisopropylphosphine as described in Example 1, method 3.

Synthesis of Methylphosphonamidite of 2′-deoxy-2-thiothymidine (2,Scheme 1):

The desired methylphoanmidite 2 is is prepared from5′-O-DMT-2′-deoxy-2-thiothymidine (Connolly et al., Nucleic Acids Res.,1989, 17, 4957) and chloro-N,N-diisopropylaminomethylphosphine asdescribed in Example 1, method 2.

Synthesis of Isopropylphosphonamidite of 2′-deoxy-2-thiothymidine (2,Scheme 1):

The desired isopropylphoanmidite 2 is prepared from5′-O-DMT-2′-deoxy-2-thiothymidine (Connolly et al., Nucleic Acids Res.,1989, 17, 4957) and dichloroisopropylphosphine as described in Example1, method 3.

Example 6

Synthesis of Methylphosphonamidite of 7-deazaadenosine (N⁶-bz, 2, Scheme1):

N⁶-Benzoyl-5′-O-(dimethoxytrityl)-7-deaza-2′-deoxyadenosine (1) ispurchased from Berry & Associates, Inc. 2434 Bishop Circle East Dexter,Mich., 48130 USA. Compound 1 is reacted withchloro-N,N-diisopropylaminomethylphosphine as described in Example 1,method 2 to obtain diastereomeric mixture of the desiredmethylphosphonamidite 2.

Synthesis of Methylphosphonamidite of 7-deazainosine (2, Scheme 1):

5′-O-DMT-7-deazainosine (1) is synthesized as reported in the literature(Seela and Klaus, Nucleic Acids Res., 1986, 14, 1825). Compound 1 isreacted with chloro-N,N-diisopropylaminomethylphosphine as described inExample 1, method 2 to obtain diastereomeric mixture of the desiredmethylphosphonamidite 2.

Synthesis of Methylphosphonamidite of 7-deazaguanosine (N²-tBu, 2,Scheme 1):

N²-isoBu-5′-O-DMT-7-deazaguanosine (1) is synthesized as reported in theliterature (Seela and Driller, Nucleic Acids Res., 1985, 13, 911).Compound 1 is reacted with chloro-N,N-diisopropylaminomethylphosphine asdescribed in Example 1, method 2 to obtain diastereomeric mixture of thedesired methylphosphonamidite 2.

Synthesis of Isopropylphosphonamidite of 7-deazaadenosine (N⁶-bz, 2,Scheme 1):

N⁶-Benzoyl-5′-O-(dimethoxytrityl)-7-deaza-2′-deoxyadenosine (1) ispurchased from Berry & Associates, Inc. 2434 Bishop Circle East Dexter,Mich., 48130 USA. Compound 1 is reacted with dichloroisopropylphosphineas described in Example 1, method 3 to obtain diastereomeric mixture ofthe desired isopropylphosphonamidite 2.

Synthesis of Isopropylphosphonamidite of 7-deazainosine (2, Scheme 1):

5′-O-DMT-7-deazainosine (1) is synthesized as reported in the literature(Seela and Klaus, Nucleic Acids Res., 1986, 14, 1825). Compound 1 isreacted with dichloroisopropylphosphine as described in Example 1,method 3 to obtain diastereomeric mixture of the desiredisopropylphosphonamidite 2.

Synthesis of Isopropylphosphonamidite of 7-deazaguanosine (N²-iBu, 2,Scheme 1):

N²-isoBu-5′-O-DMT-7-deazaguanosine (1) is synthesized as reported in theliterature (Seela and Driller, Nucleic Acids Res., 1985, 13, 911).Compound 1 is reacted with dichloroisopropylphosphine as described inExample 1, method 3 to obtain diastereomeric mixture of the desiredisopropylphosphonamidite 2.

Example 7

Separation of R and S Isomers of Alkylphosphonamidites:

A portion of the diastereomeric mixture of each of thealkylphosphonamidites obtained from Examples 1-6 is subjected tonormal-phase high-performance liquid chromatography to obtain respectiveR and S stereo isomers as described by Cormier and Plomley (J.Chromatography, A, 1994, 662, 401).

Example 8

^(a) (i) X—P(Cl)—N(iPr)₂, TEA/dichloromethane or X—P—[N(iPr)₂]₂,tetrazole/tetrazole-diisopropylammonium salt/MeCN; or X—P(Cl₂), one eq.HN(iPr)₂ followed by one 1 eq. 1 in MeCN/dichloromethane.General Synthetic Procedure of Methylphosphonamidite of α-anomeric B [4,A(N⁶-Bz), C(N⁴-Bz), G(N²—Ac) and U; R=OTBDMS, X=Me, Scheme 2):Method 1

5′-O-DMT-3′-O-TBDMS-6-N-benzoyl-α-adenosine,5′-O-DMT-3′-O-TBDMS-4-N-benzoyl-α-cytidine,5′-O-DMT-3′-O-TBDMS-2-N-acetyl-α-guanosine and5′-O-DMT-3′-O-TBDMS-α-uridine are prepared as reported by Debart et al.,(Nucleic Acids Res., 1992, 20, 1193). The protected nucleoside 3 thusobtianed is reacted with bis(diisopropylamino)methylphosphine and1H-tetrazole in acetonitrile to obtain a diastereomeric mixture of thedesired methylphosphonamidite 4 as reported in the literature (Lauritsenet al., Bioorg. Med. Chem. Lett., 2003, 13, 253). Thebis(diisopropylamino)methylphosphine reagent is obtained as reportedMoriarty et al. (J. Am. Chem. Soc., 1990, 112, 8575).

Method 2

5′-O-DMT-3′-O-TBDMS-6-N-benzoyl-α-adenosine,5′-O-DMT-3′-O-TBDMS-4-N-benzoyl-α-cytidine,5′-O-DMT-3′-O-TBDMS-2-N-acetyl-α-guanosine and5′-O-DMT-3′-O-TBDMS-α-uridine are prepared as reported by Debart et al.,(Nucleic Acids Res., 1992, 20, 1193). The protected nucleoside 3 thusobtianed is reacted with 1.2 equivalent ofchloro-N,N-diisopropylaminomethylphosphine (obtained from from ChemGenesCorporation, 33 Industrial Way, Wilmington, Mass.) in anhydrousdichloromethane (or THF) containing 3.0 equivalent ofdiisopropylethylamine obtain a diastereomeric mixture of the desiredmethylphosphonamidite 4 as reported by Sinha et al. (Nucleic Acids Res.,1994, 22, 3119).

Method 3

Methyldichlorophosphine (purchased from Aldrich) is reacted with oneequivalent of anhydrous diisopropylamine (purchased from Aldrich) indichloromethane containing three equivalent of diisopropylethylamine. Tothe resulting reaction mixture one equivalent of the appropriatelyprotected nucleoside 3 is added under constant stirring at ambienttemperature. The diastereomeric mixture of the desiredmethylphosphonamidite after standard workup is purified by flashsilicagel column chromatography to obtain pure compound 4 (Vaghefi etal., Nucleic Acids Res., 1995, 23, 3600).

Example 9

General synthetic Procedure of Isopropylphosphonamidite of α-anomeric B[4, A(N⁶-Bz), C(N⁴-Bz), G(N²—Ac) and U; R=OTBDMS, X=Me, Scheme 2):

Isopropyldichlorophosphine (purchased from Aldrich) is reacted with oneequivalent of anhydrous diisopropylamine (purchased from Aldrich) indichloromethane containing three equivalent of diisopropylethylamine. Tothe resulting reaction mixture one equivalent of the appropriatelyprotected nucleoside 3 is added under constant stirring at ambienttemperature. The diastereomeric mixture of the desiredisopropylphosphonamidite after standard workup is purified by flashsilicagel column chromatography to obtain pure compound 4 (Vaghefi etal., Nucleic Acids Res., 1995, 23, 3600).

Example 10

General Synthetic Procedure of tert-Butylphosphonamidite of α-anomeric B[4, A(N⁶-Bz), C(N⁴-Bz), G(N²—Ac) and U; R=OTBDMS, X=Me, Scheme 2):

tert-Butyldichlorophosphine (purchased from Aldrich) is reacted with oneequivalent of anhydrous diisopropylamine (purchased from Aldrich) indichloromethane containing three equivalent of diisopropylethylamine. Tothe resulting reaction mixture one equivalent of the appropriatelyprotected nucleoside 3 is added under constant stirring at ambienttemperature. The diastereomeric mixture of the desiredisopropylphosphonamidite after standard workup is purified by flashsilicagel column chromatography to obtain pure compound 4 (Vaghefi etal., Nucleic Acids Res., 1995, 23, 3600).

Example 11

Separation of R and S Isomers of Alkylphosphonamidites:

A portion of the diastereomeric mixture of each of thealkylphosphonamidites (4) obtained from Examples 8-10 is subjected tonormal-phase high-performance liquid chromatography to obtain respectiveR and S stereo isomers as described by Cormier and Plomley (J.Chromatography, A, 1994, 662, 401).

Example 12

Oligonucleotide Synthesis and Purification TABLE 17 siRNA duplexes withP-Alkylphosphonate backbone for biological assays. Seq. No NameSequence^(a) 11 Luc duplex ^(5′)CUUACGCUGAGUACUUCGAdTdT^(3′)^(3′)dTdTGAAUGCGACUCAUGAAGCU^(5′) 12 Luc sense^(5′)C*UUACGCUGAGUACUUCGAdTdT^(3′) 13 Luc sense^(5′)C*UUACGCUGAGUACUUCGAdT*dT^(3′) 14 Luc sense^(5′)C*UU*ACGCUGAGUACUUCGAdTdT^(3′) 15 Luc sense^(5′)C*UU*ACGCUGAGU*ACUUCGAdTdT^(3′) 16 Luc sense^(5′)C*UU*ACGCUGAGU*ACUUCGAdT*dT^(3′) 17 Luc sense^(5′)C*U*U*ACGCUGAGU*ACUUCGAdTdT^(3′) 18 Luc sense^(5′)C*U*U*ACGCUGAGU*ACUUCGA*dT*dT^(3′) 19 Luc sense^(5′)C*U*U*ACGCUGAGU*ACUUCG*A*dT*dT^(3′) 20 Luc sense^(5′)C*UU*AC*GC*UG*AG*UA*CU*UC*GA*dTd T^(3′) 21 Luc sense^(5′)CU*UA*CG*CU*GA*GU*AC*UU*CG*A*dT* dT^(3′) 22 Luc sense^(5′)C*U*U*A*C*G*C*U*G*A*G*U*A*C*U*U* C*G*A*dT*dT^(3′) 23 Luc sense^(5′)C#UUACGCUGAGUACUUCGAdTdT^(3′) 24 Luc sense^(5′)C#UUACGCUGAGUACUUCGAdT#dT^(3′) 25 Luc sense^(5′)C#UUACGCUGAGUACUUCGAdT*dT^(3′) 26 Luc sense^(5′)CUU#ACGCUGAGUACUUCGAdTdT^(3′) 27 Luc sense^(5′)C*UU#ACGCUGAGUACUUCGAdT*dT^(3′) 28 Luc sense^(5′)CUUACGCUGAGU#ACUUCGAdTdT^(3′) 29 Luc sense^(5′)CUU#ACGCUGAGU#ACUUCGAdT#dT^(3′) 30 Luc sense^(5′)C*UUACGCUGAGUACUUCGAdT#dT^(3′) 31 Luc sense^(5′)CUUACGCUGAGUACUUCGAdT#dT^(3′) 32 Luc sense^(5′)CUUACGCUGAGUACUUCGAdT⁺dT^(3′) 33 Luc sense^(5′)C*UUACGCUGAGUACUUCGAdT⁺dT^(3′) 34 Luc sense^(5′)CUUACGCUGAGU#ACUUCGAdT⁺dT^(3′) 35 Luc sense^(5′)CUU#ACGCUGAGU#ACUUCGAdT⁺dT^(3′) 36 Luc^(3′)dT#dTGAAUGCGACUCAUGAAGCU^(5′) antisense 37 Luc^(3′)dT^(#)dTGAAUGCGACUCAUGAAGC*U^(5′) antisense 38 Luc^(3′)dT⁺dTGAAUGCGACUCAUGAAGCU^(5′) antisense 39 Luc^(3′)dT⁺dTGAAUGCGACUCAUGAAGC*U^(5′) antisense 40 Luc^(3′)dT⁺dTGAAUGCGACUCAUGAAGC#U^(5′) antisense 41 VEGF^(5′)GCGGAUCAAACCUCACCAAdTdT^(3′) duplex^(3′)dTdTCGCCUAGUUUGGAGUGGUU^(5′) 42 VEGF^(5′)G*CGGAUCAAACCUCACCAAdTdT^(3′) sense 43 VEGF^(5′)G*CGGAUCAAACCUCACCAAdT*dT^(3′) sense 44 VEGF^(5′)G*C*GGAUCAAACCUCACCAAdT*dT^(3′) sense 45 VEGF^(5′)G*C*GGAUCAAACCUCACCAA*dT*dT^(3′) sense 46 VEGF^(5′)G*CG*GA*UC*AA*AC*CU*CA*CC*AA*dTd sense T^(3′) 47 VEGF^(5′)GC*GG*AU*CA*AA*CC*UC*AC*CA*AdT*d sense T^(3′) 48 VEGF^(5′)G*C*G*G*A*U*C*A*A*A*C*C*U*C*A*C* sense C*A*A*dT*dT^(3′) 49 VEGF^(5′)GCGGAUCAAACCUCACCAAdT#dT^(3′) sense 50 VEGF^(5′)G*CGGAUCAAACCUCACCAAdT#dT^(3′) sense 51 VEGF^(5′)GCGGAUCAAACCUCACCAAdT⁺dT^(3′) sense 52 VEGF^(5′)G*CGGAUCAAACCUCACCAAdT⁺dT^(3′) sense 53 VEGF^(3′)dT#dTCGCCUAGUUUGGAGUGGUU^(5′) antisense 54 VEGF^(3′)dT#dTCGCCUAGUUUGGAGUGGU*U^(5′) antisense 55 VEGF^(3′)dT⁺dTCGCCUAGUUUGGAGUGGUU^(5′) antisense 56 VEGF^(3′)dT⁺dTCGCCUAGUUUGGAGUGGU*U^(5′) antisense 57 VEGF^(3′)dT⁺dTCGCCUAGUUUGGAGUGGU#U^(5′) antisense 58 PTEN^(b)^(5′)CAAAUCCAGAGGCUAGCAGdTdT^(3′) ^(3′)dTdTGUUUAGGUCUCCGAUCGUC^(5′) 59PTEN ^(5′)C*AAAUCCAGAGGCUAGCAGdTdT^(3′) sense 60 PTEN^(5′)C*AAAUCCAGAGGCUAGCAGdT*dT^(3′) sense 61 PTEN^(5′)C*A*AAUCCAGAGGCUAGCAGdT*dT^(3′) sense 62 PTEN^(5′)C*AA*AU*CC*AG*AG*GC*UA*GC*AG*dTd sense T^(3′) 63 PTEN^(5′)CA*AA*UC*CA*GA*GG*CU*AG*CA*GdT*d sense T^(3′) 64 PTEN^(5′)C*A*A*A*U*C*C*A*G*A*G*G*C*U*A*G* sense C*A*G*dT*dT^(3′) 65 PTEN^(5′)CAAAUCCAGAGGCUAGCAGdT#dT^(3′) sense 66 PTEN^(5′)C#AAAUCCAGAGGCUAGCAGdT#dT^(3′) sense 67 PTEN^(5′)C*AAAUCCAGAGGCUAGCAGdT#dT^(3′) sense 68 PTEN^(5′)CAAAUCCAGAGGCUAGCAGdT⁺dT^(3′) sense 69 PTEN^(5′)C#AAAUCCAGAGGCUAGCAGdT⁺dT^(3′) sense 70 PTEN^(5′)C*AAAUCCAGAGGCUAGCAGdT⁺dT^(3′) sense 71 PTEN^(3′)dT#dTGUUUAGGUCUCCGAUCGUC^(5′) antisense 72 PTEN^(3′)dT#dTGUUUAGGUCUCCGAUCGU*C^(5′) antisense 73 PTEN^(3′)dT⁺dTGUUUAGGUCUCCGAUCGUC^(5′) antisense 74 PTEN^(3′)dT⁺dTGUUUAGGUCUCCGAUCGU*C^(5′) antisense^(a)The sense strand is written 5′ to 3′ on the top line. The antisensestrand is written 3′ to 5′ below. The oligonucleotides arephosphodiester RNA except for two 3′ deoxythymidines indicated by dT inthe sequence. dt represent cholesterol conjugation at C5 of2′-deoxyuridine and dc represent cholesterol conjugation at C5 of2′-deoxycytidine. dt represent 5β-cholanic acid conjugation at C5 of2′-deoxyuridine and dc represent#5β-cholanic acid conjugation at C5 of 2′-deoxycytidine. Scrambledsequences were generated by randomizing the sequence of the sensestrand.^(b)The PTEN sequence is identical (with the exception of the ^(3′)dTdT) on the antisense strand to that of an antisense oligonucleotidewith pharmacological activity. [M. Butler, R. A. McKay, I. J. Popoff, W.A. Gaarde, D. Witchell, S. F. Murray, N. M. Dean, S. Bhanot, B.P. Monia,Diabetes. 2002 51, 1028]*Indicates racemic or R or S methylphosphonate/methylthiophosphonatebackbone#Indicates racemic or R or Sisopropylphosphonate/isopropylthiophosphonate backbone⁺Indicates racemic or R or Stert-butylphosphonate/tert-butylthiophosphonate backboneSynthesis of Oligonucleotides

The designed RNA molecules are synthesized on a 394 ABI machine usingthe standard protocols for phosphate and phosphorothioate backbone witha slight changes in the capping step by using acetic anhydride and4-(dimethylamino)(pyridine (DMAP) as the capping reagent. Thealkylphosphonate backbone is introduced as described by Hogrefe et al.(An improved method for the synthesis and deprotection ofmethylphosphonate oligonucleotides. Methods in Molecular Biology(Totowa, N.J., United States) (1993), 20 (Protocols for Oligonucleotidesand Analogs), 143-64.). A general protocol from for synthesizingalkylphosphonate oligonucleotides is described below:

1. Wash with acetonitrile

2. Detritylate

3. Wash well with acetonitrile to dry column

4. Couple using subroutine.

-   -   a. Add monomer (phosphoanmidite 2 or 4) and activator        (5-(ethylthio)-1H-tetrazole, ETT) (monomer to activator ratio,        1:4)    -   b. Couple (same amount of time as standard amidites, or extended        or double coupling if necessary)    -   c. Oxidize immediately, with no prewash, using a        low-water-content oxidant    -   d. Wash until oxidant is rinsed away

5. Cap using acetic anhydride and DMAP

6. Wash well with acetonitrile

7. Begin cycle again

Optimum reagents and conditions as recommended by Hogrefe et al. (Animproved method for the synthesis and deprotection of methylphosphonateoligonucleotides. Methods in Molecular Biology (Totowa, N.J., UnitedStates) (1993), 20 (Protocols for Oligonucleotides and Analogs),143-64.) are used to obtain phosphonate and thiophosphonate backbonemodified oligonucleotides. Commercially available DNA and RNAphosphoramidites and supports are used unless otherwise specified.Commercial phosphoramidites with fast protecting groups(5′-O-dimethoxytritylN6-phenoxyacetyl-2′-O-t-butyldimethylsilyladenosine-3′-O—N,N′-diisopropyl-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N-4-acetyl-2′-O-t-butyldimethylsilylcytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N-2-p-isopropylphenoxyacetyl-2′-O-t-butyldimethylsilylguanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,and5′-O-dimethoxytrityl-2′-O-t-butyldimethylsilyluridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramiditeare purchased either from Pierce Nucleic Acids Technologies, Milwaukee,Wis. or from Proligo LLC, Boulder, Colo. All 2′-O-Me amidites arereceived from Glen Research. All amidites are used at a concentration of0.15 M in acetonitrile (CH₃CN) and a coupling time of 8-15 min. Theactivator is 5-(ethylthio)-1H-tetrazole (0.25M), for the PO-oxidationIodine/Water/Pyridine is used and for PS-oxidation, 2% Beaucage reagent(Iyer et al., J. Am. Chem. Soc., 1990, 112, 1253) in anhydrousacetonitrile is used. The sulphurization time is about 6 min.

Deprotection-I (Nucleobase Deprotection)

After completion of the synthesis, the support is dried thoroughly andis transferred into a screw-cap vial. The support is then mixed with asolution of absolute ethanol:acetonitrile:ammonium hydroxide (45:45:10,stored at 5° C. or freshly prepared, about 1 mL for 1 μM scalesynthesis). The suspension is vortexed for 30 min, after 30 min 1 vol ofethylenediamine is added and vortex for 6 h. The solution is decantedand the support is washed twice with acetonitrile:water (1:1). Washingsand the deprotection solution are combined and lyophilized to dryness.

Deprotection-II (Removal of 2′ TBDMS Group)

The white residue obtains is resuspended in 400 μl of triethylamine,triethylamine trihydrofluoride (TEA.3HF) and NMP (4:3:7) and heats at50° C. for overnight to remove the tert-butyldimethylsilyl (TBDMS)groups at the 2′position (Wincott et al., Nucleic Acids Res., 1995, 23,2677). The reaction is then quenched with 400 μl ofisopropoxytrimethylsilane (iPrOMe₃Si, purchased from Aldrich) andfurther incubates on the heating block leaving the caps open for 10 min;(This causes the volatile isopropxytrimethylsilylfluoride adduct tovaporize). The residual quenching reagent is removed by drying in aspeed vac. 1.5 ml of 3% triethylamine in diethyl ether is added and theoligonucleotide is pelleted out by centrifuging. The supernatant ispipetted out without disturbing the pellet and the pellet is dried inspeed vac to obtain the crude oligonucleotide as a white fluffymaterial.

Quantitation of Crude Oligomer or Raw Analysis

Samples are dissolved in RNase free deionied water (1.0 mL) andquantitates as follows: Blanking is first performed with water alone (1mL); 20 μL of sample and 980 μL of water are mixed well in a microfugetube, transfers to cuvette and absorbance reading is obtained at 260 nm.The crude material is dried down and stored at −20° C.

Purification of Oligomers:

(a) PAGE Purification

PAGE purification of oligomer synthesized is performed as reported bySambrook et al. (Molecular Cloning: a Laboratory Manual, Second Edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). A12% denaturing gel is prepared for purification of unmodified andmodified oligonucleotides. To a mixture of 120 mL Concentrate, 105 mLDiluents and 25 mL Buffer (National Diagnostics) is added 50 μL TEMEDand 1.5 mL 10% APS. After pouring the gel, it is left for ½ h topolymerize. Oligonucleotide is suspended in 20 μL water and 80 μLformamide. Loads gel tracking dye on left lane followed by the sampleslowly on to the gel. Run the gel on 1×TBE buffer at 36 W for 4-6 h.Once run is completed, transfer the gel on to preparative TLC plates andsee under UV light. Cut the bands, soak and crush in RNase free waterand leaves the vial containing purified oligonucletide in a shaker forovernight. Eluent is removed, wash residue with more RNase free water,combined washing and lyophilize to obtain the pure oligonucleotide.

Desalting of Purified Oligomer

The purified dry oligomer is desalted using Sephadex G-25 M (AmershamBiosciences). The cartridge is conditioned with 10 mL of RNase freedeionised water thrice. Finally the purified oligomer is dissolved in2.5 mL RNase free water and passed through the cartridge with very slowdrop wise elution. The salt free oligomer is eluted with 3.5 mL of RNasefree water directly into a screw cap vial.

Analysis:

Capillary Gel Electrophoresis (CGE) and electrospray LC/Ms

Approximately 0.10 OD of oligomer is first dried down, then redissolvsin water (50 □L) and pipettes in specified vials for CGE and LC/MSanalysis.

Example 13

In Vitro Cell Culture Activities of siRNA Containing Alkylphosphonate orAlkylthiophosphonate Backbone:

Dual Luciferase Gene Silencing Assays

Sense and antisense strands were arrayed into PCR tubes or plates (VWR,West Chester, Pa.) in annealing buffer (100 mM KOAc, 30 mM HEPES, 2 mMMgOAc, pH 7.4) to give a final concentration of 20 μM duplex. Annealingwas performed employing a thermal cycler (ABI PRISM 7000, AppliedBiosystems, Foster City, Calif.) capable accommodating the PCR tubes orplates. The oligoribonucleotides were held at 90° C. for two minutes and37° C. for one hour. Duplex formation was verified by native agarose gelelectrophoresis of a random sample of the sense and antisensecombinations.

HeLa SS6 cells were grown at 37° C. in Dulbecco's modified Eagle medium(DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/mLpenicillin, and 100 μg/mL streptomycin (Invitrogen, Carlsbad, Calif.).HeLa Dual-luc cells (HeLa cells stably expressing both firefly andrenilla luciferase) were grown at 37° C. in Eagle medium supplementedwith 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100μg/mL streptomycin, 0.5 μg/mL puromycin, 500 μg/mL zeocin (Invitrogen,Carlsbad, Calif.). Cells were passaged regularly to maintain exponentialgrowth. Twenty-four hours prior to siRNA transfection, cells were seededon opaque, white 96-well plates (Costar, Corning, N.Y.) at aconcentration of 15,000 cells/well in antibiotic-free, phenol red-freeDMEM (Invitrogen).

In vitro activity of siRNAs was determined using a high-throughput96-well plate format luciferase silencing assay. Assays were performedin one of two possible formats. In the first format, HeLa SS6 cells werefirst transiently transfected with plasmids encoding firefly (target)and renilla (control) luciferase. DNA transfections were performed usingLipofectamine 2000 (Invitrogen) and the plasmids gWiz-Luc (Aldevron,Fargo, N. Dak.) (200 ng/well) and pRL-CMV (Promega, Madison, Wis.) (200ng/well). After 2 h, the plasmid transfection medium is removed, and thefirefly luciferase targeting siRNAs were added to the cells at variousconcentrations. In the second format, HeLa Dual-luc cells (stablyexpressing both firefly and renilla luciferase) are directly transfectedwith firefly luciferase targeting siRNAs. SiRNA transfections wereperformed using either TransIT-TKO (Mirus, Madison, Wis.) orLipofectamine 2000 according to manufacturer protocols. After 24 h,cells were analyzed for both firefly and renilla luciferase expressionusing a plate luminometer (VICTOR², PerkinElmer, Boston, Mass.) and theDual-Glo Luciferase Assay kit (Promega). Firefly/renilla luciferaseexpression ratios were used to determine percent gene silencing relativeto mock-treated (no siRNA) controls. TABLE 18 Methylphosphonate backboneand Luc activity Seq. Luc No Sequence^(a) Activity 101^(5′)CUUACGCUGAGUACUUCGAdTdT^(3′) Active^(3′)dTdTGAAUGCGACUCAUGAAGCU^(5′) 102 ^(5′)CUUACGCUGAGUACUUCGAdT*dT^(3′)Active ^(3′)dTdTGAAUGCGACUCAUGAAGCU^(5′) 103^(5′)CUUACGCUGAGUACUUCGAdTdT^(3′) Active^(3′)dT*dTGAAUGCGACUCAUGAAGCU^(5′) 104^(5′)CUUACGCUGAGUACUUCGAdT*dT^(3′) Active^(3′)dT*dTGAAUGCGACUCAUGAAGCU^(5′)*MethylphosphonateSingle incorporation of methylphosphonate linkages at the 3′-end ofsense and antisense retain luciferase activity (Table 18).

Example 14

Serum Stability of siRNAs Containing Alkylphosphonate orAlkylthiophosphonate Backbone:

siRNA duplexes were prepared at a stock concentration of 1 μM in whicheither the sense (S) or antisense strand (AS) contains a trace amount of5′-³²P labeled material (e.g. ³²P-S/AS and S/³²P-AS). The presence ofthe end-labeled sense or antisense strand allows for monitoring of theindividual strand within the context of the siRNA duplex. Therefore, twoduplex preparations are made for each siRNA sequence tested. siRNAduplexes were incubated in 90% human serum at a final concentration of100 nM duplex. Samples were removed and quenched in a stop mix atappropriate times. For a typical time course, 10 seconds, 15 minutes, 30minutes, 1 hour, 2 hours and 4 hours time points are taken. Samples wereanalyzed by denaturing polyacrylamide gel electrophoresis along with acontrol sample (4 hour buffer-alone incubation) and a partial alkalinehydrolysis ladder of the labeled sense or antisense strand as a marker.The gel is imaged using a Fuji phosphorimager to detect the full lengthsense and antisense strands along with any degradation fragments thatare generated by serum nucleases during incubation.

Serum stability of methylphosphonate backbone modification was testedand the result showed enhanced serum stability as compared to aunmodified siRNA duplex. A description of each modification, itslocation within the siRNA duplex, and the serum stability data follows.

The results from denaturing gel analysis of the human serum stabilityassay for duplex 101 and 104 (Table 18) are presented in FIG. 13. C isthe 4 hour time point for siRNA duplex incubated in PBS buffer alone,OH⁻ is the partial alkaline hydrolysis marker, *s/as represents siRNAduplex containing 5′ end-labeled sense RNA and s/*as represents duplexcontaining 5′ end-labeled antisense RNA. Samples were incubated in 90%human serum and time points were assayed at 10 seconds, 5 min, 15 min,30 min, 1 hour, 2 hours, and 4 hours. Black lines to the right of bandsindicate exonucleolytic degradation fragments and the red lineshighlight a few of the endonucleolytic degradation fragment.Methylphosphonate substitution at the 3′ end inhibit exonucleasedegradation of the 3′ overhangs.

The parent duplex used to establish a serum stability baseline forevaluating the effects of chemical modifications on nuclease resistanceis shown in FIG. 13. Duplex 101 (Table 18) was subjected to the serumstability assay to evaluate its inherent nuclease resistance and todefine its degradation pattern (FIG. 13, duplex 101). This unmodifiedduplex is degraded by both 3′-5′ exonucleases and endonucleases.

Cleavage of the 3′ end of both the sense and antisense strands by 3′-5′exonucleases occurs within the first 5 minutes of incubation resultingin the loss of the 3′ terminal dT residues (black line in FIG. 13,duplex 101). In addition to exonuclease degradation, both strands arecleaved by endonucleases. There is a major endonuclease site at positionsixteen of the antisense strand (red line in FIG. 13, duplex 101) thatappears as early as 10 seconds. Very little full length sense orantisense strand is remaining after 1 hour in human serum.

Specific phophodiester linkages of the siRNA duplex were replaced bymethylphosphonate and their stability was evaluated in the human serumstability assay (FIG. 13, duplex 104). Substitution of thephosphodiester linkage at the 3′ end of both the sense and antisensestrands inhibits exonucleolytic degradation of the 3′ overhangs (FIG.13, duplex 104) as compared to the unmodified parent duplex 101. Fulllength starting material is present out to four hours for both the senseand antisense strands. The endonucleolytic cleavage pattern seen in theunmodified duplex is unchanged. In summary, a single methylphosphonatebetween the two 3′ terminal nucleotides is sufficient to protect the 3′ends from exonuclease degradation.

METHODS

Method 1

Binding Affinity of siRNA Containing Alkylphosphonate orAlkylthiophosphonate Backbone to Plasma Proteins.

Measurement of Binding Affinity:

To measure binding affinity of siRNAs to plasma protein, the 5′ end ofthe sense strand of an siRNA duplex is labeled with ³²P using T4polynucleotide kinase using standard procedures. Each of the siRNAduplexes shown in Table I will be tested in this assay. Theunincorporated label is removed using a G25 column and labeling isconfirmed by polyacrylamide gel electrophoresis. A fixed concentrationof labeled RNA (50 nM) and complementary strand (50 nM) is incubatedwith increasing concentration of plasma proteins at 25° C. for one hourin phosphate-buffered saline buffer containing 0.1 mM EDTA and 0.005%Tween 80. After incubation, the samples are loaded onto low binding,regenerated cellulose filter membranes with a molecular weight cut-offof 30,000 (Millipore). The samples are spun gently in a microfuge(NYCentrifuge 5415C; Eppendorf, Westbury, N.Y.) at 3000 rpm (735 g) for3 to 6 minutes, allowing collection of −20% of the loaded volume in thefiltrate.

Radioactivity present in aliquots from the filtrate and the initial(unfiltered) solutions is measured using a scintillation counter (modelLS6000IC, Beckman, Fullerton, Calif.). The counts obtained in thefiltrate aliquots represent the free (unbound) RNA, and appropriatecalculations are performed to obtain the concentration of free RNA.Further calculations yield the concentration of RNA bound to protein.See R. Zini, J. Barre, F. Bree, J. P. Tillement, B. Sebille, J.Chromatogr. 1981, 216, 191 and A. N. Kuznetsov, G. V. Gyul'khandanyan,B. Ebert, Mol. Biol. (Moscow) 1977, 11, 1057.

The extent of siRNA binding to plasma proteins is determined using anequilibrium filtration method. The fraction of bound RNA is plotted vs.the total protein concentration. The equilibrium constant, K_(d), isdetermined from nonlinear regression analysis of the fraction of siRNAbound (f_(bound)) as a function of the free protein concentration(f_(free)). Thus, the data can be fit to a two-state model:

where O is the unbound siRNA, A is the unbound protein, OA is thesiRNA-protein complex and K_(A) is the equilibrium association constant.Method 2Inhibition of mRNA Expression in Balb-C Mouse Treated with siRNAs.Female BALB/c mice (6 weeks old, Harlan Sprague Dawley, Indianapolis,Ind.) are housed three to a cage under conditions meeting NationalInstitue of Health regulations (19). siRNAs, including unconjugated andscrambled controls and vehicle containing no siRNA are administered in0.9% NaCl, i. p. at indicated dose levels once daily for three days andtissues are harvested for analysis.

Total mRNA is extracted from mouse liver by rapid homogenization of thetissue in 4 M guanidinuim isothiocyanate followed by centrifugation overa cesium chloride gradient. RNAs (20-40 μg) are resolved in 1.2% agarosegels containing 1.1% formaldehyde and transferred to nylon membranes.The blots are hybridized with a radiolabelled human cDNA probe. Probeshybridized to mRNA transcripts are visualized and quantified using aPhosPhorImager (Molecular Dynamics). After stripping the blots ofradiolabelled probe, they are reprobed with G3PDH cDNA to confirm equalloading.

Method 3

siRNA Treatment of Human Tumor Cells in Nude Mice-IntraperitonealInjection. Human lung carcinoma A549 cells are harvested and 5×10⁶ cells(200 μL) were injected subcutaneously into the inner thigh of nude mice.Palpable tumors develop in approximately one month. siRNAs that targetthe c-raf and the H-ras messages, including steroid/lipid-conjugated RNAand scrambled controls and vehicle containing no siRNA are administeredto mice intraperitoneally at a dosage of 20 mg/kg body weight, everyother day for approximately ten weeks. Mice are monitored for tumorgrowth during this time.

Method 4

siRNA Treatment of Human Breast Tumor Cells in Nude Mice. Human breastcarcinoma MDA-MB-231 cells are harvested and 5×10⁵ cells (200 μL) areinjected subcutaneously into the mammary fat pads of athymic nude mice.Palpable tumors develop in approximately one month. siRNAs that targetthe c-raf and the H-ras messages, including steroid/lipid-conjugatedsiRNA and scrambled controls and vehicle containing no siRNA areadministered to mice intraperitoneally at a dosages of 5, 10, and 25mg/kg/day body weight, every day for approximately 20 days. Mice aremonitored for tumor growth during this time.

Method 5

siRNA Treatment of Human Lung Tumor Cells in Nude Mice. Human lungcarcinoma A549 cells are harvested and 5×10⁶ cells (200 μL) are injectedsubcutaneously into the inner thigh of nude mice. Palpable tumorsdevelop in approximately one month. siRNAs that target the c-raf and theH-ras messages, including cholesterol or cholanic acid—conjugated RNAand scrambled controls and vehicle containing no siRNA are administeredto mice subcutaneously at the tumor site. Drug treatment begins one weekfollowing tumor cell inoculation and is given twice a week for fourweeks. Mice are monitored for tumor growth for a total of nine weeks.

Method 6

Inhibition of Apo-B mRNA Expression in Hep G-2 cells and in Balb-C MouseTreated with siRNAs. Inhibition of Aop-B mRNA expression by siRNA (TableI, siRNAs 141-146) will be evaluated in vitro and in vivo. Effect ofsiRNA treatment on message levels in HEP-G2 cells is analyzed followingtreatment. The procedure is described by Yao Z Q, Zhou Y X, Guo J, FengZ H, Feng X M, Chen C X, Jiao J Z, and Wang S Q in “Inhibition ofhepatitis B virus in vitro by antisense oligonucleotides.” Acta Virol.1996, 40(1), 35-9.

Female BALB/c mice (6 weeks old, Harlan Sprague Dawley, Indianapolis,Ind.) are housed three to a cage under conditions meeting NationalInstitue of Health regulations (19). siRNAs, including unconjugated andscrambled controls and vehicle containing no siRNA are administered in0.9% NaCl, i. p. at indicated dose levels once daily for three days andtissues are harvested for analysis.

Total mRNA is extracted from mouse liver by rapid homogenization of thetissue in 4 M guanidinuim isothiocyanate followed by centrifugation overa cesium chloride gradient. RNAs (20-40 μg) are resolved in 1.2% agarosegels containing 1.1% formaldehyde and transferred to nylon membranes.The blots are hybridized with a radiolabelled human Apo-B cDNA probe asdescribed (20). Probes hybridized to mRNA transcripts are visualized andquantified using a PhosPhorImager (Molecular Dynamics). After strippingthe blots of radiolabelled probe, they are reprobed with G3PDH cDNA toconfirm equal loading.

Example 15

Synthesis of Naproxen-Bearing Tether.

(i) DCC, DMAP, DIEA/Dichloromethane; (ii) LiOH/THF—H₂O; (iii) DCC, DMAP,Pentafluorophenol/Dichloromethane; (iv) Serinol, TEA/Dichloromethane;(v) DMT-C1, DMAP/Py; (vi) (a) Succinic anhydride, DMAP/Dichloroethaneand (b)) DTNP, DMAP, Ph₃P, Aminoalkyl solid support and (vii)N,N-diisopropylamino b-cyanoethylphosphonamidic chloride{[(CH₃)₂CH]₂N—P(Cl)—OCH₂CH₂CN}, DIEA/Dichloromethane or2-Cyanoethyl-N,N,N′,N′-tetraisopropylphosphane, tetrazole (ortetrazolediisopropylammonium salt)/Acetonitrile.

N-Naproxyl-6-aminohexanoic Acid Methyl Ester (3a):

The ester 3a was prepared according to reported procedure from theliterature (Org. Syn., 1984, 63, 183). Naproxen (1a 10.00 g, 43.427mmol, purchased from Aldrich) and 4-(Dimethylamino)pyridine (DMAP, 0.53g, 4.338 mmol, purchased from Aldrich) were dissolved in anhydrousN,N-dimethylformamide (DMF) and 1,3-diisopropylcarbodiimide (DICC, 6.8mL, 43.914 mmol, purchased from Aldrich) was added into the solution andstirred at ambient temperature for 5 minute. 6-aminohexanoic acid methylester hydrochloride (2, 10.00 g, 57.408 mmol, purchased from Fluka) anddiisopropylethylamine (DIEA, 10 mL, purchased from Aldrich) were addedinto the stirring solution after 5 minute of addition of DICC andstirred overnight at ambient temperature. DMF was removed from thereaction in vacuo, the product was extracted into ethyl acetate (EtOAc,200 mL), washed successively with aqueous KHSO₄, water, sodiumbicarbonate solution and water. The organic layer was dried overanhydrous sodium sulfate (Na₂SO₄) and filtered. A white solid wasprecipitated out from the EtOAc extract by adding hexane to afford thedesired compound 3a, 11.20 g (72.14%). ¹H NMR (400 MHz, [D₆]DMSO, 25°C.): δ 7.95-7.92 (t, J(H,H)=5.2 & 5.6 Hz, 1H), 7.76-7.68 (m, 3H),7.43-7,40 (dd, J′(H,H)=1.6 and J″(H,H)=8.4 Hz, 1H), 7.25-7.24 (d,J(H,H)=2.0 Hz, 1H), 7.13-7.11 (dd, J′(H,H)=2.4 and J″(H,H)=8.8 Hz, 1H),3.84 (s, 3H), 3.70-3.65 (q, J(H,H)=6.8 and 7.2 Hz, 1H), 3.54 (s, 3H),3.00-2.97 (q, J(H,H)=6.8 Hz, 2H), 2.21-2.17 (t, 2H), 1.48-1.29 (m, 7H),1.19-1.13 (m, 2H).

N-Naproxyl-6-aminohexanoic Acid (4a):

Hydrolysis of the ester 3a was performed as reported earlier (Rajeev etal., 2002, 4, 4395). Compound 3a (10.80 g, 30.24 mmol) was suspended intetrahydrofuran-water (THF—H₂O) mixture (4:1, 40 mL) and stirred withLiOH (1.65 g, 39.32 mmol) for 4 h at ambient temperature. THF wasremoved from the reaction in vacuo and free acid was precipitated outfrom water by adding concentrated KHSO₄ solution, thoroughly washed withwater, filtered through a sintered filter, triturated with diethyl etherand dried over P₂O₅ under vacuum overnight to obtain the acid 4a as awhite solid, 10.22 g (98.4%). ¹H NMR (400 MHz, [D₆]DMSO, 25° C.): δ11.96 (bs, 1H), 7.95-7.92 (t, J(H,H)=5.37 Hz, 1H), 7.77-7.68 (m, 3H),7.43-7.41 (d, J(H,H)=8.3 Hz, 1H), 7.25-7.24 (d, J(H, H)=2.44 Hz, 1H),7.13-7.11 (dd, J′(H,H) 1.95, 2.44 and J″(H,H)=8.79, 9.27 Hz, 1H), 3.84(s, 3H), 3.71-3.65 (q, J(H,H)=6.84, 7.33 Hz, 1H), 3.02-2.97 (m, 2H),2.13-2.09 (t, J(H,H)=7.33 Hz, 2H), 1.46-1.30 (m, 7H), 1.21-1.15 (m, 2H).

N-Naproxyl-6-aminohexanoic Acid Pentafluorophenyl Ester (5a):

Compound 4a (5.00 g, 14.57 mmol), DMAP (0.18 g, 1.47 mmol) andpentafluorophenol (3.50 g, 19.02 mmol, purchased from Aldrich) weretaken in dichloromethane (40 mL) and DCC (3.00 g, 14.54 mmol) was addedinto the solution. Reaction mixture was stirred at ambient temperaturefor 8 h. The reaction mixture was diluted to 100 mL by adding EtOAc andprecipitated DCU was removed by filtration. Combined filtrate,evaporated solvent in vacuo, and the residue was subsequently filteredthrough a column of silica gel, eluent hexane/EtOAc 4:1 to obtain amixture (7.90 g) of the desired ester 5a and excess pentafluorophenolfrom the reaction. The crude product thus obtained was directly used forproceeding experiments without further purification.

Naproxen-6-aminohexanoic Acid—Serinol Conjugate (6a)

Pentafluorophenol ester 5a was stirred with serinol in the presence ofTEA to obtain compound 6a (J. Org. Chem., 1991, 56, 1713). Compound 5a(4.00 g, 7.86 mmol) and serinol (1.5 g, 16.46 mmol, purchased fromAldrich) were suspended in dichloromethane (30 mL) and triethylamine(TEA, 2.3 mL, purchased from Aldrich) was added into the suspension,stirred at ambient temperature for 2 h. A white precipitate was formedduring the course of the reaction. After 2 h, the precipitate wasfiltered through a sintered filter, washed successively with excess ofdichloromethane, water and diethyl ether to afford desired product 6a(2.82 g, 86.2%). ¹H NMR (400 MHz, [D₆]DMSO, 25° C.): δ 7.95-7.92 (t,J(H, H)=5.49 Hz, 1H, exchangeable with D₂O), 7.77-7.68 (m, 3H),7.43-7.39 (m, 2H, accounted for 1H after D₂O exchange), 7.26-7.25 (d,J(H,H)=2.14 Hz, 1H), 7.13-7.11 (dd, J′(H,H)=2.44 and J″(H,H)=8.85 Hz,1H), 4.58-4.55 (t, J(H,H)=5.49 Hz, 2H, exchangeable with D₂O), 3.84 (s,3H), 3.71-3.65 (m, 2H), 3.37-3.35 (t, became doublet after D₂O exchange,4H), 3.02-2.95 (m, 2H), 2.03-2.01 (t, J(H,H)=7.32, 7.63 Hz, 2H),1.46-1.30 (m, 7H), 1.20-1.12 (m, 2H).

Naproxen-6-aminohexanoic Acid—Serinol Mono DMT (7a)

Compound 6a was prepared by modifying reported literature procedure(Rajeev et al., Org. Lett., 2003, 5, 3005). A solid mixture of compound6a (2.50 g, 6.01 mmol) and DMAP (0.075 g, 0.61 mmol) was dried over P₂O₅under vacuum overnight. The solid mixture was suspended in anhydrouspyridine (100 mL) under argon and heated to obtain a homogenoussolution. The temperature of the mixture was brought to room temperatureand stirred. 4,4′-Di-O-methyltrityl chloride (2.24 g, 6.61 mmol,purchased from Chem Genes Corporation) was separately dissolved in 20 mLof anhydrous dichloromethane and added drop-wise into the stirringpyridine solution over a period of 45 minute under argon. Reactionmixture was further stirred overnight. Solvents were removed form thereaction mixture and the product was extracted into EtOAc (150 mL) andwashed successively with water, NaHCO₃ solution and water, dried overanhydrous Na₂SO₄ and evaporated to solid mass. Desired product waspurified by flash silica gel column chromatography: (a) eluent: 1%methylalcohol (MeOH) in dichloromethane—1.60 g of undesired bis DMTderivative (26.1%) and (b) 5% MeOH in dichloromethane—2.50 g of desiredproduct 7a (57.9%). ¹H NMR (400 MHz, [D₆]DMSO, 25° C.): δ 7.94-7.91 (t,J(H,H)=5.49 Hz, 1H, exchangeable with D₂O), 7.7-7.68 (m, 3H), 7.60-7.58(d, J(H,H)=8.55 Hz, 1H, exchangeable with D₂O), 7.43-7.10 (m, 12H),6.86-6.84 (d, 4H), 4.62-4.59 (t, J(H,H)=5.18, 5.49 Hz, 1H, exchangeablewith D₂O), 4.01-3.96 (m, 1H), 3.83 (s, 3H), 3.71-3.65 (m, 7H), 3.44-3.42(t, J(H,H)=5.19, 5.49 Hz, 2H), 3.03-2.87 (m, 4H), 2.05-2.01 (t,J(H,H)=7.33, 7.63 Hz, 2H), 1.48-1.30 (m, 7H), 1.21-1.14 (m, 2H).

Naproxen-6-aminohexanoic Acid—Serinol CPG (8a)

The desired solid support 8a was prepared according to reportedprocedures (References for succinilation: Rajeev et al., Org. Lett.,2003, 5, 3005 and for conjugation to CPG: Kumar et al., NucleosidesNucleotides, 1996, 15, 879). A mixture of compound 7a (1.00 g, 1.39mmol), succinic anhydride (0.17 g, 1.69 mmol, purchased from Aldrich)and DMAP (0.21 g, 1.72 mmol) were suspended in 7 mL of anhydrousethylene dichloride for 24 h. Reaction mixture was diluted to 50 mL byadding dichloromethane and washed with dilute aqueous citric acidsolution (20 mL), dried over anhydrous Na₂SO₄ and evaporated to dryness.The residue obtained was further dried over P₂O₅ under vacuum to affordan almost pure but crude monosuccinate as a white solid (1.10 g, 96.5%).The product obtained was directly used for subsequent reaction withoutfurther purification. ¹H NMR (400 MHz, [D₆]DMSO, 25° C.): δ 7.94-7.91(t, J(H,H)=5.19, 5.49 Hz, 1H, exchangeable with D₂O), 7.83-7.81 (d,J(H,H)=7.94 Hz, 1H, exchangeable with D₂O), 7.76-7.68 (m, 3H), 7.42-7.10(m, 12H), 6.88-6.86 (d, 4H), 4.18-4.12 (m, 2H), 4.07-3.98 (m, 2H), 3.83(s, 3H), 3.71-3.66 (m, 7H), 3.00-2.91 (m, 4H), 2.40 (s, 4H), 2.04-2.00(t, J(H,H)=7.32 Hz, 2H), 1.44-1.22 (m, 7H), 1.19-1.15 (m, 2H).

2,2′-Dithiobis(5-nitropyridine) (0.38 g, 1.22 mmol, purchased fromAdrich) was dissolved in a 1:1 mixture of acetonitrile and ethylenedichloride (5 mL) and added into a suspension ofnaproxen-6-aminohexanoic acid—serinol conjugate mono DMT mono succinate(1.00 g, 1.21 mmol) and DMAP (0.16 g, 1.31 mmol) in 2 mL of anhydrousacetonitrile. Triphenylphosphine (Ph₃P, 0.32 g, 1.22 mmol, purchasedfrom Aldrich) was added into the reaction mixture and shaken for 3-4minute. 5.5 g of long chain aminoalkyl controlled-pore-glass (CPG) with500 size and a loading of 112.7 μM/g (purchased from Millipore), andexcess of acetonitrile (to soak the CPG completely) were added into thereaction mixture and the suspension was shaken (agitated) for 45 minuteat ambient temperature. CPG was filtered through a sintered funnel,washed extensively with acetonitrile, dichloromethane and diethyl etherand subsequently re-suspended in pyridine-dichloromethane and treatedwith acetic anhydride in the presence of DIEA to cap unreacted aminogroups on the CPG. After 10 minute, CPG was filtered and extensivelywashed with dichloromethane, acetonitrile and diethyl ether followed bydrying under vacuum to obtain the desired CPG 8a with a loading 54.12μM/g. The loading was determined as reported in the literature (Prakashet al., J. Org. Chem., 2002, 67, 357 and references cited therein).

Naproxen-6-aminohexanoic Acid—Serinol mono DMT Phosphoramidite (9a).

The phosphoramidite was prepared as reported in the literature (Rajeevet al., Org. Lett., 2003, 5, 3005 and references cited therein).Compound 7a (1.00 g, 1.39 mmol) and diisopropylammonium tetrazolide(0.12 g, 0.70 mmol) were dried over P₂O₅ vacuum overnight andsubsequently suspended in anhydrous acetonitrile (5 mL) under argonatmosphere. 2-Cyanoethyl-N,N,N′,N′-tetraisopropylphosphane (0.69 mL,2.09 mmol) was added into the suspension and stirred at ambienttemperature for 14 h. Solvent was removed form the reaction in vacuo andresidue was suspended in EtOAc (40 mL) and washed with dilute NaHCO₃solution followed by standard work. Desired amidite 9a was purified byflash silica gel column chromatography; eluent: EtOAc, yield 0.79 g(61.8%). ³¹P NMR (161.8 MHz, CDCl₃, 25° C.): δ 146.01, 145.69.

Naproxen Pentafluropehenol Ester (1c):

Naproxen (1, 11.25 g, 48.86 mmol), pentafluorophenol (10.00 g, 54.33mmol) and DMAP (0.60 g, 4.91 mmol) were dissolved in DMF (40 mL) andstirred at ambient temperature. 1,3-dicyclohexylcarbodiimide (DCC, 11.00g, 53.31 mmol) was added into the solution and continued stirringovernight. 1,3-dicyclohexylurea (DCU) was precipitated out during thecourse of the reaction. The precipitated DCU was filtered off, washedwith DMF, combined filtrate and removed DMF in vacuo. Oily residueobtained was filtered through a small column of silica gel, eluent 10%EtOAc in hexane to remove dissolved DCU to afford a mixture of thedesired ester 1c and excess pentafluorophenol (20.30 g). The crudeproduct thus obtained was directly used for proceeding experimentswithout further purification. ¹H NMR (400 MHz, [D₆]DMSO, 25° C.): δ7.85-7.81 (m, 3H), 7.48-7.46 (dd, J′(H,H)=1.53 and J″(H,H)=8.55 Hz, 1H),7.32-7.31 (d, J(H,H)=2.44 Hz, 1H), 7.18-7.16 (dd, J′(H,H)=2.44 andJ″(H,H)=8.85 Hz, 1H), 4.47-4.44 (q, J(H,H)=7.02 Hz), 3.86 (s, 3H),1.63-1.61 (d, J(H,H)=7.34 Hz, 3H).

Example 16

Synthesis of Ibuprofen-Bearing Tether.

(i) DCC, DMAP, DIEA/Dichloromethane; (ii) LiOH/THF—H₂O; (iii) DCC, DMAP,Pentafluorophenol/Dichloromethane; (iv) Serinol, TEA/Dichloromethane;(v) DMT-Cl, DMAP/Py; (vi) (a) Succinic anhydride, DMAP/Dichloroethaneand (b) DTNP, DMAP, Ph₃P, Aminoalkyl solid support and (vii)N,N-diisopropylamino b-cyanoethylphosphonamidic chloride{[(CH₃)₂CH]₂N—P(Cl)—OCH₂CH₂CN}, DIEA/Dichloromethane or2-Cyanoethyl-N,N,N′,N′-tetraisopropylphosphane, tetrazole (ortetrazolediisopropylammonium salt)/Acetonitrile.N-Ibuprofyl-6-aminohexanoic Acid Methyl Ester (3b):

Ibuprofen (1b, 5.0 g, 24.23 mmol, purchased from Acros Organic), methyl6-aminohexanoic acid monohydrochloride (2, 6.60 g, 36.33 mmol, purchasedfrom Fluka) and DMAP (0.30 g, 2.46 mmol) were suspended indichloromethane (60 mL) in a 200 mL round bottom flask and DCC (5.00 g,24.23 mmol) was added into the suspension, stirred for 3 minute. After 3minute, 3.6 mL (25.83 mmol) of TEA was added into the reaction andcontinued stirring at ambient temperature for 18 h. Solvent and excessTEA were removed from the reaction in vacuo and residue obtained wastriturated with diethyl ether, filtered through a sintered funnel toremove DCU. Combined filtrate and evaporated on a rotary evaporator.Residue was redissolved in EtOAc (100 mL) and successively washed withKHSO₄ solution, water, NaHCO₃ solution and water followed by drying overanhydrous Na₂SO₄ and evaporation of solvent in vacuo to obtain yellowishviscous residue of compound 3b (8.0 g). The crude product thus obtainedwas directly used for subsequent reaction without further purification.¹H NMR (400 MHz, [D₆]DMSO, 25° C.): δ 7.86-7.84 (bt, J(H,H)=5.39, 5.00Hz, 1H, exchangeable with D₂O), 7.19-7.03 (m, 4H), 3.56 (s, 3H),3.53-3.47 (q, J(H,H)=7.05 Hz, 1H), 3.00-2.95 (q, J(H,H)=6.64, 5.81 Hz,2H), 2.39-2.37 (m, 2H, mixture of rotamers), 2.23-2.20 (t, J(H1,H)=7.45,7.05 Hz, 2H), 1.81-1.74 (m, 1H), 1.49-1.41 (m, 2H), 1.36-1.26 (m, 5H),1.19-1.11 (m, 2H), 0.84-0.82 (m, 6H, mixture of rotamers).

N-Ibuprofyl-6-aminohexanoic Acid (4b):

Compound 3b (8.00 g, 24.01 mmol) was stirred with LiOH (1.21 g, 28.84mmol) in THF—H₂O (4:1, 40 mL) for 4 h. Solvents were removed from thereaction mixture in vacuo and the residue was washed with concentratedKHSO₄ solution. Unlike the corresponding naproxen analogue 4a, the freeacid 4b did not precipitate out from the aqueous phase, so the aqueousphase was repeatedly extracted with EtOAc, combined extract, dried overNa₂SO₄ and evaporated in vacuo to obtain slightly yellowish viscousresidue, 6.60 g (86.1%). The acid 4b thus obtained was directly used forsubsequent experiments without further purification. ¹H NMR (400 MHz,[D₆]DMSO, 25° C.): δ 11.96 (bs, 1H, exchangeable with D₂O), 7.87-7.84(t, J(H,H)=5.39 Hz, 1H, exchangeable with D₂O), 7.19-7.04 (m, 4H),4.04-3.99 (q, J(H,H)=7.05 Hz, 1H), 3.62-3.57 (q, J(H,H)=7.05 Hz, 0.1H,minor rotamer), 3.53-3.47 (q, J(H,H)=7.05 Hz, 1.9H), 3.00-2.95 (q,J(H,H)=6.22 Hz, 2H), 2.41-2.37 (m, 2H, mixture of rotamers), 2.14-2.10(t, J(H,H)=7.47, 7.05 Hz, 2H), 1.81-1.74 (m, 1H), 1.46-1.40 (m, 2H),1.36-1.26 (m, 5H), 1.20-1.12 (m, 2H), 0.85-0.82 (m, 6H, mixture ofrotamers).

N-Ibuprofyl-6-aminohexanoic Acid Serinol Conjugate (6b):

Compound 4b (6.60 g, 20.676 mmol), DMAP (0.26 g, 2.128 mmol) andpentafluorophenol (5.70 g, 30.97 mmol) were dissolved in dichloromethane(60 mL) and DCC (4.27 g, 20.70 mmol) was added into the stirringsolution. The reaction mixture was allowed to stir for 8 h. PrecipitatedDCU was removed by filtration and the filtrate was evaporated to obtaina crude oil containing the desired ester 5b. The crude 5b thus obtainedwas stirred with serinol (3.5 g, 38.42 mmol) in dichloromethane in thepresence of TEA (8 mL) for 2 h. A white precipitate was formed duringthe course of the reaction, which was filtered washed successively withdichloromethane, water and diethyl ether and dried over P₂O₅ to obtain2.4 g of the product 6b. Extraction of the aqueous phase with EtOAcafforded another 1.05 g of the desired product 6b. Combined yield was42.5%. ¹H NMR (400 MHz, [D₆]DMSO, 25° C.): δ 7.87-7.84 (t, J(H,H)=5.86,5.37 Hz, 1H, exchangeable with D₂O), 7.42-7.40 (d, J(H,H)=7.81 Hz, 1H,exchangeable with D₂O), 7.19-7.17 (d, J(H,H)=8.30 Hz, 2H), 7.06-7.04 (d,J(H,H)=8.30 Hz, 2H), 4.57 (bs, 2H, exchangeable with D₂O), 3.69-3.63 (m,1H), 3.53-3.47 (q, J(H,H)=6.83 Hz, 1H), 3.36-3.34 (d, J(H,H)=5.37 Hz,4H), 3.02-2.91 (m, 2H), 2.39-2.37 (d, J(H,H)=7.34 Hz, 2H), 2.04-2.00 (t,J(H,H)=7.33 Hz, 2H), 1.81-1.75 (m, 1H), 1.44-1.26 (m, 7H), 1.18-1.12 (m,2H), 0.84-0.83 (d, J(H,H)=6.35 Hz, 6H).

N-Ibuprofyl-6-aminohexanoic Acid Serinol Mono DMT (7b):

A solid mixture of compound 6b (3.00 g, 7.65 mmol), 4,4′-dimethoxytritylchloride (2.85 g, 8.41 mmol) and DMAP (0.20 g, 1.64 mmol) was taken in a200 mL RB and dried over P₂O₅ under vacuum overnight. Anhydrous pyridine(40 mL) was added into the mixture under argon and stirred forovernight. Pyridine was removed from the reaction and residue wassuspended in EtOAc (100 mL) followed by standard workup. Desired monoDMT and bis DMT products were separated by flash silica gel columnchromatography, eluent: 2-3% methanol in dichloromethane, 170 g (22.3%,bis DMT derivative) and eluent: 4% methanol in dichloromethane, 1.89 g(35.6%, desired mono DMT product 7b). ¹H NMR (400 MHz, [D₆]DMSO, 25°C.): δ 7.83-7.80 (t, J(H,H)=5.37 Hz, 1H, exchangeable with D₂O),7.58-7.55 (d, J(H,H)=8.79 Hz, 1H, exchangeable with D₂O), 7.34-7.32 (d,J(H,H)=7.33 Hz, 2H), 7.26-7.14 (m, 9H), 7.02-7.00 (d, J(H,H)=7.81 Hz,2H), 6.83-6.81 (d, J(H,H)=8.79 Hz, 4H), 4.58-4.56 (t, J(H,H)=5.37, 4.88Hz, 1H, exchangeable with D₂O), 3.95-3.93 (m, 1H), 3.68 (s, 6H),3.48-3.45 (q, J(H,H)=7.34 Hz, 1H), 3.41-3.38 (t, J(H,H)=5.37 Hz, 2H),2.96-2.84 (m, 4H), 2.34-2.33 (d, J(H,H)=7.33 Hz, 2H), 2.02-1.98 (t,J(H,H)=7.33, 7.81 Hz, 2H), 1.76-1.69 (m, 1H), 1.44-1.36 (m, 2H),1.33-1.23 (m, 5H), 1.16-1.08 (m, 2H), 0.80-0.78 (d, J(H,H)=6.35 Hz, 6H).¹³C NMR (100 MHz, [D₆]DMSO, 25° C.): δ 174.0, 172.8, 158.3, 145.4,139.9, 139.7, 136.2, 130.1, 129.2, 128.2, 128.1, 127.3, 113.5, 85.5,61.0, 55.4, 51.1, 45.1, 44.6, 35.7, 30.0, 29.1, 26.3, 25.4, 22.5, 18.8.

Ibuprofen-6-aminohexanoic Acid—Serinol CPG (8b):

The desired succinate (0.98 g, 85.7%) was synthesized from thecorresponding precursor 7b (1.00 g, 1.44 mmol), DMAP (0.27 g, 2.21 mmol)and succinic anhydride (0.22 g, 2.20 mmol) as described for thecorresponding naproxen derivative. The succinic acid derivative waspurified by flash silica gel column chromatography, eluent: 5% methanolin dichloromethane. ¹H NMR (400 MHz, [D₆]DMSO, 25° C.): δ 7.86-7-80 (m,2H, exchangeable with D₂O), 7.34-7.32 (d, J(H,H)=7.33 Hz, 2H), 7.28-7.13(m, 9H), 7.02-7.00 (d, J(H,H)=8.30 Hz, 2H), 6.85-6.83 (d, J(H,H)=8.79Hz, 4H), 4.14-1.10 (bm, 2H), 4.02-3.98 (m, 1H), 3.68 (s, 6H), 3.50-3.44(q, J(H,H)=7.33, 6.83 Hz, 2H), 2.96-2.87 (m, 2H), 2.35-2.33 (m, 6H),2.51-2.45 (m, 7H, 2H+DMSO-d₆), 2.01-1.96 (t, J(H,H)=7.32 Hz, 2H),1.77-1.69 (m, 1H), 1.42-1.22 (m, 7H), 1.15-1.07 (m, 2H), 0.80-0.78 (d,J(H,H)=6,35 Hz, 6H). ¹³C NMR (100 MHz, [D₆]DMSO, 25° C.): δ 174.9,174.3, 173.2, 158.5, 145.3, 139.9, 139.8, 136.0, 130.2, 129.3, 128.4,128.1, 127.4, 113.6, 85.8, 55.5, 46.1, 46.1, 45.3, 44.7, 35.6, 30.1,29.0, 26.2, 25.4, 22.6, 18.8.

The desired CPG 8b (4.50 g) with a loading capacity of 85.62 μM/g wasprepared from 0.92 g (1.16 mmol) of the ibuprofen succinate thusobtained, 2,2′-Dithiobis(5-nitropyridine) (0.37 g, 1.18 mmol), DMAP(0.15 g, 1.23 mmol), Ph₃P (0.31 g, 1.18 mmol) and long chain aminoalkylcontrolled-pore-glass (CPG) with 500 size and a loading of 162.5 μM/g asdescribed for the preparation of the corresponding naproxen analogue 8a.

Ibuprofen Pentafluorophenol Ester (1d):

Ibuprofen pentafluorophenol ester (1d) was prepared from ibuprofen (1b,5.00 g, 24.23 mmol), pentafluorophenol (5.4 g, 29.02 mmol), DCC (5.00 g,24.23 mmol) and DMAP (0.30 g, 2.46 mmol) as described for the synthesisof pentafluorophenol ester (1c) of naproxen (1a).

Example 17

Synthesis of Naproxen-Bearing Linker

^(a) (i) N-hydroxysuccinimide, DCC, DMAP/Dichloromethane-DMF; (ii)Pentafluorophenol, DCC, DMAP/Dichloromethane; (iii) Serinol,TEA/dichloromethane; (iv) DMT-Cl, DMAP/Py; (v) Pd—C (10%), ammoniumformate; (vi) Naproxen-NHS ester (14), TEA/Dichloromethane; (vii)Ibuprofen-NHS ester (15), TEA/Dichloromethane.N-Cbz-6-aminohexanoic Acid Pentafluorophenol Ester 11b:

N-Cbz-6-aminohexanoic acid (10, 30.31 g, 114.25 mmol, purchased fromNovabiochem), pentafluorophenol (25.00 g, 135.83 mmol) and DMAP (1.54 g,12.60 mmol) were taken in dichloromethane (100 mL) and to this DCC(26.00 g, 121.01 mmol) added slowly under stirring. During the course ofaddition, temperature of the reaction rose and dichloromethane startedboiling out, so it was cooled down to room temperature and allowed tostir overnight. Reaction mixture was diluted to 200 mL by adding diethylether and subsequently filtered through a sintered funnel to remove DCU,washed residue with diethyl ether, combined washing and evaporated todryness. The desired product 11b was purified by flash silica gel columnchromatography, eluent: hexane/EtOAc 2:1, yield 43.54 g (88.4%). ¹H NMR(400 MHz, [D₆]DMSO, 25° C.): δ 7.36-7.23 (m, 6H), 4.99 (s, 2H),3.01-2.96 (q, J(H,H)=6.35 Hz, 2H), 2.78-2.52 (q, J(H,H)=7.33 Hz, 2H),1.69-1.61 (m, 2H), 1.47-1.29 (m, 4H).

N-Cbz-6-aminohexanoic Acid Serinol (12):

Compound 11b (26.00 g, 60.31 mmol) and serinol (5.00 g, 54.88 mmol) weresuspended in 200 mL of dichloromethane and stirred in the presence ofTEA (17 mL, 121.97 mmol) at ambient temperature overnight. A thick whiteprecipitate was formed during the course of the reaction. The reactionmixture was diluted to 200 mL by adding diethyl ether, triturated andfiltered. The precipitate was thoroughly washed with diethyl ether anddried under vacuum over P₂O₅ to obtain 16.51 g (81.0%) of the desiredcompound 12 as a white solid. ¹H NMR (400 MHz, [D₆]DMSO, 25° C.): δ7.44-7.42 (d, J(H,H)=7.81 Hz, 1H, exchangeable with D₂O), 7.37-7.27 (m,5H), 7.24-7.20 (t, J(H,H)=5.86, 5.37 Hz, 1H, exchangeable with D₂O),4.99 (s, 2H), 4.58-4.55 (t, J(H,H)=5.37 Hz, 2H, exchangeable with D₂O),3.70-3.65 (m, 1H), 3.37-3.34 (t, J(H,H)=5.86, 3.37 Hz, changed todoublet after D₂O exchange, J(H,H, after D₂O exchange)=5.37 Hz, 4H),2.98-2.92 (q, J(H,H)=6.84, 6.35 Hz, 2H), 2.06-2.02 (t, J(H,H)=7.33 Hz,2H), 1.49-1.33 (m, 4H), 1.24-1.16 (m, 2H).

N-Cbz-6-aminohexanoic Acid Serinol Mono DMT (13):

Compound 12 (14.10 g, 41.66 mmol) and DMAP (0.60 g, 4.91 mmol) weretaken in a 200 mL RB and dried under vacuum over P₂O₅. The solid mixturethen suspended in 50 mL of anhydrous pyridine under argon.4,4-Dimethoxytrityl chloride (15.5 g, 44.27 mmol) was separatelydissolved in 40 mL of anhydrous dichloromethane and added into thestirring pyridine solution under argon. The reaction mixture was allowedto stir at ambient temperature overnight. Solvents were removed from thereaction mixture and residue was extracted into EtOAC (200 mL), washedwith NaHCO₃ solution followed by standard workup. The desired product 13was purified by flash silica gel column chromatopgraphy, eluent:hexane/EtOAc 3:2, 8.62 g (28.0%, bis DMT derivative) and 3-4% MeOH inchloroform, 15.28 g (57.3%, desired mono DMT derivative 13). ¹H NMR (400MHz, [D₆]DMSO, 25° C.): δ 7.63-7.60 (d, J(H,H)=8.79 Hz, 1H, exchangeablewith D₂O), 7.38-7.17 (m, 15H, accounted for 14H after D₂O exchange),6.87-6.84 (d, J(H,H)=8.79 Hz, 4H), 4.98 (s, 2H), 4.62-4.59 (t,J(H,H)=5.37 Hz, 1H, exchangeable with D₂O), 4.00-3.95 (m, 1H), 3.72 (s,6H), 3.46-3.41 (t, J(H,H)=5.37 Hz, 2H), 3.00-2.87 (m, 4H), 2.08-2.04 (t,J(H,H)=7.33 Hz, 2H) 1.50-1.33 (m, 4H), 1.25-1.16 (m, 2H).

Synthesis of Compound 7a from Compound 13

DCC (14.80 g, 71.73 mmol) was added into a stirring mixture of naproxen(15.00 g, 65.14 mmol), DMAP (0.80 g, 6.55 mmol) and N-hydroxysuccinimide(10.00 g, 86.82 mmol) in 80 mL of DMF at ambient temperature and thestirring was continued overnight. Precipitated DCU was filtered off fromthe reaction, washed with DMF, combined the washings and evaporated todryness in vacuo. Residue obtained was triturated with diethyl ether,filtered, washed the residue extensively with diethyl ether and dried toobtain a white solid naproxen N-hydroxy succinimide ester, 22.00 g(21.31 g theoretical value). ¹H NMR (400 MHz, [D₆]DMSO, 25° C.): δ7.83-7.78 (m, 3H), 7.46-7.43 (dd, J′(H,H)=1.95, 1.46 and J″(H,H)=8.79,8.30 Hz, 1H), 7.32-7.31 (d, J(H,H)=2.44 Hz, 1H), 7.17-7.15 (dd,J′(H,H)=2.44 and J″(H,H)=8.79 Hz, 1H), 4.41-4.35 (q, J(H,H)=6.84, 7.33Hz, 1H), 3.86 (s, 3H), 2.74 (s, 4H), 1.59-1.57 (d, J(H,H)=6.84 Hz, 3H).

Compound 13a:

Compound 13 and ammonium formate are suspended in a 1:1 mixture ofmethanol-EtOAc and 10% by wt Pd—C (10%) is added into the suspension,the reaction mixture is slightly warmed using a heat gun and allowedstir at ambient temperature for 2 h. Removed Pd—C and insoluble ammoniumformate by filtration, combined filtrate and evaporated. Residue wassuspended in EtOAc and washes with aqueous NaHCO₃ solution to obtaincompound 13a.

Synthesis of Compound 7a from Compound 13

Naproxen N-hydroxysuccinimide ester (21.0 g) was prepared from naproxen(1a, 15.00 g, 65.14 mmol) and N-hydroxysuccinimide (10.00 g, 86.82 mmol)using DCC (14.80 g, 71.73 mmol) as the coupling agent in the presence ofDMAP (0.80 g, 6.55 mmol) as described in Example 15 for the synthesis ofthe corresponding pentafluorophenol ester 1c. ¹H NMR (400 MHz, [D₆]DMSO,25° C.): δ 7.83-7.78 (m, 3H), 7.46-7.43 (dd, J′(H,H)=1.95, 1.46 andJ″(H,H)=8.79, 8.30 Hz, 1H), 7.32-7.31 (d, J(H,H)=2.44 Hz, 1H), 7.17-7.15(dd, J′(H,H)=2.44 and J″(H,H)=8.79 Hz, 1H), 4.41-4.35 (q, J(H,H)=6.84,7.33 Hz, 1H), 3.86 (s, 3H), 2.74 (s, 4H), 1.59-1.57 (d, J(H,H)=6.84 Hz,3H).

Naproxen N-hydroxysuccinimide ester is stirred with compound 13a toobtain compound 7a. See Example 15 for analytical data.

Synthesis of Compound 7b from Compound 13

DCC (6.60 g, 31.99 mmol) was added into a stirring mixture of ibuprofen(6.00 g, 29.09 mmol), DMAP (40 g, 3.27 mmol), and N-hydroxysuccinimide(4.40 g, 38.23 mmol) in 30 mL of DMF and allowed to stir overnight. DCUwas filtered off as described for the synthesis of the correspondingnaproxen derivative. Residue obtained was triturated with diethyl etherand filtered, the product dissolved in ether. Combined filtrate, reducedto small volume on the rotary evaporator. Hexane was added into theconcentrated to solution to precipitate out the desired product, whichwas filtered, washed with hexane and dried to obtain the desired ester7.48 g, (yield 84.8%). ¹H NMR (400 MHz, [D₆]DMSO, 25° C.): δ 7.28-7.25(d, J(H,H)=8.30 Hz, 2H), 7.16-7.13 (d, J(H,H)=8.30 Hz, 2H), 4.24-4.18(q, J(H,H)=6.84, 7.32 Hz, 1H), 2.77 (s, 4H), 2.43-2.41 (d, J(H,H)=7.32Hz, 2H), 1.84-1.77 (m, 1H), 1.49-1.47 (d, J(H,H)=7.32 Hz, 3H), 0.85-0.83(d, J(H,H)=6.84 Hz, 6H).

Naproxen N-hydroxysuccinimide ester is stirred with compound 13a toobtain compound 7b. See Example 16 for analytical data.

Example 18

Synthesis of Naproxen Bound to a Solid Support

(i) TEA/Dichloromethane; (ii) (a) LiBH₄/MeOH and (b) DMT-Cl, DMAP/Py;(iii) (a) Succinic anhydride, DMAP/Dichloroethane and (b) DTNP, DMAP,Ph₃P, Aminoalkyl solid support and (iv) N,N-diisopropylaminob-cyanoethylphosphonamidic chloride {[(CH₃)₂CH]₂N—P(Cl)—OCH₂CH₂CN},DIEA/Dichloromethane or 2-Cyanoethyl-N,N,N′,N′-tetraisopropylphosphane,tetrazole (or tetrazolediisopropylammonium salt)/Acetonitrile.Compound 6c:

Compound 5a (2.90 g, 5.70 mmol) and commercially availabletrans-4-hydroxy-L-proline methyl ester hydrochloride (1.25 g, 6.88 mmol,obtained from CNH Technologies Inc.) were suspended in dichloromethane(30 mL) and excess TEA was added into the suspension and stirred atambient temperature for 2 h. Solvent and excess TEA were removed fromthe reaction mixture in vacuo and the product was extracted into EtOAc(100 mL). The organic layer was successively washed with aqueous KHSO₄solution, water, NaHCO₃ solution and water followed by standard workup.Residue obtained was purified by flash slice gel column chromatography,eluent 5% MeOH in dichloromethane, to afford compound 6c, 2.1 g (78.4%).¹H NMR (400 MHz, [D₆]DMSO, 25° C.): δ 7.94-7.92 (bt, 1H, exchangeablewith D₂O), 7.77-7.68 (m, 3H), 7.43-7.41 (dd, J′(H,H)=1.66, 1.40 andJ″(H,H)=8.30 Hz, 1H), 7.26-7.25 (d, J(H,H)=2.10 Hz, 1H), 7.14-7.11 (dd,J′(H,H)=2.49 and J″(H,H)=8.71 Hz, 1H), 5.16-5.15 (d, 0.85H, exchangeablewith D₂O, major rotamer), 5.09-5.08 (d, 0.15H, exchangeable with D₂O,minor rotamer), 4.59-4.57 (m, 0.15H), 4.30-4.20 (m, 1.85H), 3.84 (s,3H), 3.69-3.52 (m, 6H including H₂O from the solvent), 3.34-3.32 (bd,2.5H, accounted for 1H after D₂O exchange), 3.02-2.97 (m, 2H), 2.15-2.04(m, 3H), 1.89-1.82 (m, 1H), 1.44-1.30 (m, 7H), 1.15-1.14 (m, 2H). ¹³CNMR (100 MHz, [D₆]DMSO, 25° C.): δ 173.9, 173.0, 171.7, 157.3, 137.8,133.4, 129.4, 128.7, 126.9, 126.7, 125.5, 118.9, 106.0, 69.1, 57.5,55.5, 55.0, 52.1, 45.5, 38.7, 33.7, 29.0, 26.1, 24.1, 18.7.

Compound 7c:

Compound 6c is treated with LliBH₄ in methanol to obtain thecorresponding diol (Rajeev et al., J. Org. Chem., 1997, 62, 5169). Thediol thus obtained is stirred with DMT-Cl in anhydrous pyridine in thepresence of DMAP to obtain compound 7c.

Solid Support 8c:

The desired CPG 8c is prepared from compound 7c as described in Example15 for the synthesis of compound 8a.

Phosphoramidite 9c:

The desired phosphoramidite 9c is prepared from compound 7c as describedin Example 15 for the synthesis of compound 9a

Example 19

Synthesis of Naproxen Bound to a Solid Support

(i) Ethyl glyoxalate, NaBH(OAc)₃, HOAc/MeOH; (ii) Ethyl bromopropionate,DIEA/Dichloromethane; (iii) KO^(t)Bu/Toluene: (iv) Baker's yeast/H₂O;(v) LiBH₄/MeOH; (vi) DMT-Cl, DMAP/Py; (vii) (a) Succinic anhydride,DMAP/Dichloroethane and (b) DTNP, DMAP, Ph₃P, Aminoalkyl solid supportand (viii) N,N-diisopropylamino b-cyanoethylphosphonamidic chloride{[(CH₃)₂CH]₂N—P(Cl)—OCH₂CH₂CN}, DIEA/Dichloromethane or2-Cyanoethyl-N,N,N′,N′-tetraisopropylphosphane, tetrazole (ortetrazolediisopropylammonium salt)/Acetonitrile.Compound 15:

Compound 14 is prepared as reported in the literature. General procedurefor synthesizing amine 15 (Ref: Abdel-Magid et al. J. Org. Chem. 1996,61 (11), 3849-3862): A representative example of this reductiveamination is shown with the reaction of amine 14 and ethyl glyoxalate:Ethylglyoxalate (45% solution in Toluene; 1 equiv.) and amine (1 equiv.)are mixed in anhydrous THF and then treated with sodiumtriacetoxyborohydride (1.5 equiv.). The mixture is stirred at ambienttemperature for 24 h. The reaction mixture is quenched by the additionof saturated NaHCO₃ solution and the product is extracted into EtOAc.Amine 15 is obtained by the concentration of organic layer.

Compound 16:

Synthesis of diester 16 (Ref: St-Denis et al. Can. J. Chem. 2000, 776):To a solution of freshly prepared amine 15 (1 equiv.) in toluene isadded ethyl 3-bromopropionate (1.2 equiv) in toluene. The suspension isheated at 60° C. for 6 h and poured into aqueous sodium carbonatesolution. The aqueous phase is extracted with chloroform andconcentrated to afford diester 16.

Compound 17 and 18:

Synthesis of ketoester 17 and 18 (Ref: Blake et al. J. Org. Chem. 1964,5293)

To a suspension of potassium t-butoxide (1.5 equiv) in toluene at 0° C.under nitrogen is added 1 equiv. of diester 16 in toluene over a periodof 30 min. The solution is stirred at 0° C. till the starting materialdisappears and glacial acetic acid is added, immediately followed by asolution of NaH₂PO₄.H₂O in ice-cold water. The resultant mixture isextracted with chloroform and the combined organic extracts are washedtwice with pH 7.0 phosphate buffer, dried and evaporated to a residue.The residue is dissolved in toluene, cooled to 0° C., and extracted withportions of cold pH 9.5 carbonate buffer. The aqueous extract isconverted to pH 3 with slow addition of phosphoric acid and extract withchloroform (3×100 mL). The combined organic layer is dried overanhydrous sodium sulfate and evaporated to afford ketoester 18. Toluenelayer is dried over sodium sulfate and evaporated to dryness to yieldketoester 17.

Compound 19:

Baker's yeast reduction of 18 to obtain compound 19 (Ref: St-Denis etal. Can. J. Chem. 2000, 776). To a solution of sucrose (2 equiv by wt.)in distilled water is added baker's yeast (1.5 equiv. by wt.). Thesuspension is heated at 32° C. in the rotary evaporator. The content ofthe flask is then poured into diester 18 (1 equiv. by wt.). Stirring iscontinued for a day after which additional sucrose in warm (40° C.)distilled water is added. After 2 days Celite is added to the mixtureand is filtered through a sintered glass funnel. The filtrate isre-filtered through a pad of Kieselguhr. After washing, the aqueouslayer is extracted with dichloromethane. The organic layer is combinedand evaporated to yield ester alcohol 19.

Diol 20:

Compound 19 (1 equiv.) is dissolved in anhydrous THF and is added to 1Mlithium borohydride (1 Equiv.) in anhydrous THF at 0° C. The reactionmixture is stirred at 0° C. till the disappearance of startingmaterials. Excess lithium borohydride is quenched by the addition ofwater. The reaction mixture is concentrated under reduced pressure. Tothe residue 3N hydrochloric acid is added and stirred for 3 h. Theresultant aqueous layer is extracted with ethyl acetate. The combinedorganic layer is dried over sodium sulfate and concentrated to yielddiol 20 which is purified by column chromatography.

Compound 21:

Compound 21 is obtained from the diol 20 as described in Example 15 forthe preparation of compound 7a from diol 6a.

Solid Support 22:

Compound 22 is obtained from compound 21 as described in Example 15 forthe preparation of compound 8a from compound 7a.

Phosphoramidite 23:

Phosphoramidite 22 is obtained from compound 21 as described in Example15 for the preparation of compound 9a from compound 7a.

Example 20

Synthesis of Naproxen Bound to a Solid Support

(i) DMT-Cl, DMAP/Py; (ii) (a) Piperidine/DMF (b) 11a,TEA/Dichloromethane (iii) H₂/Pd—C or Ammonium formate, Pd—C; (iv) 1c,TEA/Dichloromethane (v) DL-6-Methoxy-α-methyl-2-napthalenemethanol,CDI/THF (vi) (a) Succinic anhydride, DMAP/Dichloroethane and (b) DTNP,DMAP, Ph₃P, Aminoalkyl solid support and (vii) N,N-diisopropylaminob-cyanoethylphosphonamidic chloride {[(CH₃)₂CH]₂N—P(Cl)—OCH₂CH₂CN},DIEA/Dichloromethane or 2-Cyanoethyl-N,N,N′,N′-tetraisopropylphosphane,tetrazole (or tetrazolediisopropylammonium salt)/Acetonitrile.Compound 25:

Compound 24 is prepared as reported in the literature (Filichev andPedersen, Tetrahedron, 2001, 57, 9163-68). The mono DMT compound 25 isprepared from compound 24 as described in Example 15 for the preparationof compound 7a from compound 6a.

Compound 26:

Fmoc group is removed from compound 25 by treating with piperidine asreported in the literature (Atherton and Sheppard, The Peptides, 1987,9, 1, Udenfriend and Meienhofer Eds., Academic Press, New York). Afterremoving Fmoc, the free amine obtained is stirred with compound 11a (seeScheme 2) in the presence of TEA to obtain compound 26.

Compound 27:

Catalytic hydrogenation of compound 26 yields compound 27.

Compound 28:

Compound 27 is stirred with the ester 1c (Scheme 1) in the presence ofTEA to obtain compound 28.

Compound 29:

The phosphoramidite 29 is prepared from compound 28 as described inExample 15 for the preparation of phosphoramidite 9a from 7a.

Compound 30:

The solid support 30 is obtained from 28 as described in Example 15 forthe preparation of support 8a from 7a.

Compound 31:

Compound 27 is treated with 1,1′-carbonyldiimidazole and commerciallyavailable DL-6-Methoxy-α-methyl-2-napthalenemethanol (Acros Organics) inTHF as reported in the literature to obtain compound 31 (Hernandez andHodges, J. Org. Chem., 1997, 62, 3153).

Compound 32:

The phosphoramidite 32 is prepared from compound 31 as described inExample 15 for the preparation of compound 9a from compound 7a.

Compound 33:

The solid support 33 is obtained from compound 31 as described for thepreparation of support 8a from compound 7a.

Example 21

Synthesis of Naproxen Bound to a Solid Support

(i) TBDMS-Cl, Imidazole/Pyridine; (ii) H₂/Pd—C; (iii) for 36a,DL-6-methoxy-α-methyl-2-Naphthalenemethanamine, CDI/THF and for 36b,DL-6-Methoxy-α-methyl-2-napthalenemethanol, CDI/THF; (iv) (a) Succinicanhydride, DMAP/Dichloroethane and (b) DTNP, DMAP, Ph₃P, Aminoalkylsolid support and (v) N,N-diisopropylamino b-cyanoethylphosphonamidicchloride {[(CH₃)₂CH]₂N—P(Cl)—OCH₂CH₂CN}, DIEA/Dichloromethane or2-Cyanoethyl-N,N,N′,N′-tetraisopropylphosphane, tetrazole (ortetrazolediisopropylammonium salt)/Acetonitrile.Compound 34

Compound 13 (12.91 g, 20.16 mmol) was stirred with TBDMS-Cl (4.60 g,30.52 mmol) in pyridine in the presence of imidazole (6.30 g, 92.54mmol) at ambient temperature under argon for 6 h. Pyridine was removedfrom the reaction mixture in vacuo and residue was extracted into EtOAc(100 mL) and washed with NaHCO₃ solution followed by standard workup.Residue was purified by flash silica gel column chromatography to obtaincompound 34, eluent: 2-3% methanol in dichloromethane, yield: 15.10 g(99.3%). ¹H NMR (400 MHz, [D₆]DMSO, 25° C.): δ 7.65-7.63 (d, J(H,H)=8.30Hz, 1H, exchangeable with D₂O), 7.38-7.17 (m, 15H, accounted for 14Hafter D₂O exchange), 6.86-6.84 (d, J(H,H)=8.79 Hz), 4.01-3.96 (m, 1H),3.71 (s, 6H), 3.58-3.54 (m, 2H), 3.04-2.88 (m, 4H), 2.08-2.04 (t,J(H,H)=7.33 Hz, 2H), 1.49-1.31 (m, 4H), 1.23-1.17 (m, 2H), 0.72 (s, 9H),−0.08 (s, 3H), −0.10 (s, 3H).

Compound 35:

Compound 34 is stirred with ammonium formate and Pd—C to obtain compound35 as described in Example 17 for the preparation of compound 13a fromcompound 13.

Compound 36a:

Compound 35 is treated with 1,1′-carbonyldiimidazole andDL-6-methoxy-α-methyl-2-Naphthalenemethanamine as described in Example20 for the preparation of compound 31 from compound 27.DL-6-methoxy-α-methyl-2-Naphthalenemethanamine is prepared according toliterature procedure (Wolber and Ruechardt, Chem. Ber., 1991, 124,1667). After making the completely protected urea derivative, theproduct obtained is treated with TEA.3HF (Nystrom et. al., TetrahedronLett., 1985, 26, 5393) in the presence of excess of TEA in THF to obtaincompound 36a.

Compound 36b:

Compound 35 is treated with 1,1′-carbonyldiimidazole and commerciallyavailable DL-6-Methoxy-α-methyl-2-napthalenemethanol (Acros Organics) asdescribed in Example 20 for the preparation of compound 31 from compound27. After making the completely protected carbamte derivative, theproduct obtained is treated with TEA.3HF (Nystrom et al., TetrahedronLett., 1985, 26, 5393) in the presence of excess of TEA in THF to obtaincompound 36b.

Compound 37a

The phosphoramidite 37a is prepared from compound 36 as described inExample 15 for the preparation of phosphoramidite 9a from 7a.

Compound 38a:

The solid support 38a is obtained from 36a as described in Example 15for the preparation of support 8a from 7a.

Compound 37b:

The phosphoramidite 37b is prepared from compound 36b as described inExample 15 for the preparation of compound 9a from compound 7a.

Compound 38b:

The solid support 38b is obtained from compound 36b as described for thepreparation of support 8a from compound 7a.

Example 22

Synthesis of Naproxen Bound to a Solid Support

Compound 42a:

Compound 39 was purchased from Chem Genes Corporation. Compound 39 (1.50g, 2.15 mmol) and compound 1c (1.30 g, 3.28 mmol, see Example 15 for thepreparation of 1c) were stirred in dichloromethane (10 mL) in thepresence of excess TEA for 4h. The reaction mixture was diluted after to80 mL by adding more dichloromethane and washed with NaHCO₃ solution,the organic layer was evaporated to dryness. Residue obtained waspurified by flash silica gel column chromatography to afford compound42a (0.85 g, 43.5%, eluent: 4% MeOH in dichloromethane). ¹H NMR (400MHz, [D₆]DMSO, 25° C.): δ 11.61 (s, 1H, exchangeable with D₂O),8.00-7.91 (bm, 3H, partly exchangeable with D₂O), 7.76-7.68 (m, 3H),7.43-7.01 (m, 15H), 6.87-6.83 (m, 4H), 6.17-6.14 (t, J(H,H)=6.41, 6.71Hz, 1H), 5.28-5.27 (d, J(H,H)=4.88 Hz, 1H, exchangeable with D₂O),4.23-4.19 (m, 1H), 3.89-3.82 (m, 4H), 3.71-3.65 (m, 8H), 3.32 (s, 3H),3.32-2.90 (m, 6H), 2.49-2.31 (m, 1H), 2.29-2.13 (m, 1H), 1.38-1.18 (m,9H).

Compound 43a:

3′-O-succinate (0.67 g, 92.8%) of compound 42a (0.65 g, 0.71 mmol) wasprepared as described in Example 15. ¹H NMR (400 MHz, [D₆]DMSO, 25° C.):δ 12.19 (bs, 1H, exchangeable with D₂O), 11.64-11.60 (bm, 1H,exchangeable with D₂O), 8.03-7.75 (m, 3H), 7.76-7.67 (m, 3H), 7.42-7.01(m, 15H), 6.87-6.76 (m, 4H), 6.16-6.12 (t, J(H,H)=6.71, 7.02 Hz, 1H),5.17-5.15 (m, 1H), 4.08-3.99 (m, 2H), 3.84-3.82 (m, 3H), 3.71-3.65 (m,9H), 3.30-3.19 (m, 2H), 3.11-2.98 (m, 6H), 2.65-2.40 (11H), 2.34-2.28(m, 1H), 1.37-1.28 (m, 9H), 1.17-1.13 (m, 10H).

The 3′-O-succinate (0.51 g, 0.50 mmol) thus obtained was conjugated toCPG as described in example 15 for the preparation of compound 8a toobtain the desired CPG. Loading 12 μM/g, was determined as described inthe literature (Prakash et al., J. Org. Chem., 2002, 67, 357 andreferences cited therein).

Example 23

In Vitro Luc Activity of siRNA TABLE 19 In vitro Luc activity of siRNAwith methyl- phosphonate backbone at terminal and internal positions invitro Purity Luc Mass (%, Activ- Sequence Calc. Found CGE) ity^(c)101^(a) 5′ CUUACGCUG 6606.0 6606.45 99.2 +++ AGUACUUCGA dTd T 3′ 3′dTGAAUGCGAC 6693.3 6693.0 89.01 UCA UGAAGCU 5′ 105^(b) 5′ C*UpdTACGCU6616.20 6612.24 90.19 +++ GAGpdTACUUCGAp dTdT 3′ 3′ dTdTGAAUGCG 6693.36693.0 89.00 ACUCAUGAAGCU 5′^(a)Control Luc sequence,^(b)modified sense strand with methylphosphonate backbone and dT, and^(c)both control and modified siRNA showed comparable in vitro genesilencing.The synthesis details of the sequences in Table 19 are provided inExample 12. The details of the luciferase activity assay are provided inExample 13; the control duplex 101 is listed above in Table 18, entry101.

INCORPORATION BY REFERENCE

All of the patents and publications cited herein are hereby incorporatedby reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A compound represented by formula I:

wherein R¹ is optionally substituted aralkyl, —Si(R⁷)₃, —C(O)R⁴, —CO₂R⁴,or —C(O)(NR⁸)R⁴; R² represents independently for each occurrence H,alkyl, or halogen; R³, R⁴, and R⁷ each represent independently for eachoccurrence alkyl, aryl, or aralkyl; R⁵ is —Si(R⁷)₃, —C(O)R⁴, —CO₂R⁴, or—C(O)(NR⁸)R⁴;

R⁸ represents independently for each occurrence H, alkyl, aryl, oraralkyl; R⁹ represents independently for each occurrence H or alkyl; andthe stereochemical configuration at any stereocenter of a compoundrepresented by I is R, S, or a mixture of these configurations.
 2. Thecompound of claim 1, wherein said compound is represented by formula Ia:


3. The compound of claim 2, wherein R⁵ is Si(R⁷)₃, and R³, R⁴, and R⁷are alkyl.
 4. The compound of claim 2, wherein R⁵ is Si(R⁷)₃, and R³,R⁴, and R⁷ are alkyl; R¹ is optionally substituted dimethoxytrityl; andR⁶ is


5. The compound of claim 2, wherein R² is H, R³ is methyl, R⁴ isisopropyl, R⁵ is Si(CH₃)₂-tert-butyl, R¹ is optionally substituteddimethoxytrityl, and R⁶ is


6. The compound of claim 2, wherein R² is H, R³ is methyl, R⁴ isisopropyl, R⁵ is Si(CH₃)₂-tert-butyl, R¹ is

and R⁶ is


7. A double-stranded oligonucleotide comprising a first strand and asecond strand, wherein said first strand and said second strand arerepresented independently by formula II:

wherein X¹ is H, —P(O)(OM)₂, —P(O)(OM)-O—P(O)(OM)₂, —P(O)(Oalkyl)₂,—P(O)(Oalkyl)-O—P(O)(Oalkyl)₂, or -A⁶-[A⁷-(A⁵)_(w)]_(y); M representsindependently for each occurrence an alkali metal or a transition metalwith an overall charge of +1; R¹ and R⁵ represent independently for eachoccurrence H, alkyl, or halogen; R² and R³ represent independently foreach occurrence H, OH, F, —Oalkyl, —Oallyl, —O(C(R¹⁹)₂)_(k)OR¹⁹,—O(C(R¹⁹)₂)_(k)SR¹⁹, —O(C(R¹⁹)₂)_(k)N(R¹⁹)₂, —O(C(R¹⁹)₂)_(k)C(O)N(R¹⁹)₂,—N(R¹⁹)₂, —S(C₁-C₆)alkyl, —O(C(R¹⁹)₂)_(k)O(C₁-C₆)alkyl,—O(C(R¹⁹)₂)_(k)S(C₁-C₆)alkyl,—O(C(R¹⁹)₂)_(k)O(C(R¹⁹)₂)_(k)N((C₁-C₆)alkyl)₂,—O(C(R¹⁹)₂)_(k)ON((C₁-C₆)alkyl)₂, or —O-A⁶-[A⁷-(A⁵)_(w)]_(y); R⁴represents independently for each occurrence H, OH, F, —Oalkyl, —Oallyl,—O(C(R¹⁹)₂)_(k)OR¹⁹, —O(C(R¹⁹)₂)_(k)SR¹⁹, —O(C(R¹⁹)₂)_(k)N(R¹⁹)₂,—O(C(R¹⁹)₂)_(k)C(O)N(R¹⁹)₂, —N(R¹⁹)₂, —S(C₁-C₆)alkyl,—O(C(R¹⁹)₂)_(k)O(C₁-C₆)alkyl, —O(C(R¹⁹)₂)_(k)S(C₁-C₆)alkyl,—O(C(R¹⁹)₂)_(k)O(C(R¹⁹)₂)_(k)N((C₁-C₆)alkyl)₂, or—O(C(R¹⁹)₂)_(k)ON((C₁-C₆)alkyl)₂; R⁶, R⁷, and R⁹ represent independentlyfor each occurrence H, alkyl, aryl, or aralkyl; R⁸ representsindependently for each occurrence alkyl, aryl, or aralkyl; k representsindependently for each occurrence 1, 2, 3, or 4; n¹ is 1, 2, or 3; n² isan integer in the range of about 15-28, inclusive; w representsindependently for each occurrence 1, 2, or 3 in accord with the rules ofvalence; x represents independently for each occurrence 0, 1, 2, or 3; yrepresents independently for each occurrence 1, 2, 3, 4, or 5 in accordwith the rules of valence; A¹ represents independently for eachoccurrence:

A² represents independently for each occurrence:

A³ represents independently for each occurrence

A⁴ represents independently for each occurrence a bond, alkyl diradical,heteroalkyl diradical, alkenyl diradical, alkynyl diradical,alkylalkynyl diradical, aminoalkyl diradical, thioether, —C(O)—, —S(O)—,—S(O)₂—, B¹C(R)₂B², B¹C(R)(B²)₂, B¹C(B²)₃, B¹N(R)(B²), B¹N(B²)₂, or hasthe formula:

B¹ is a bond between A³ and A⁴; B² is a bond between A⁴ and A⁵; Rrepresents independently for each occurrence hydrogen or alkyl; mrepresents independently for each occurrence 1, 2, 3, 4, 5, 6, 7, or 8;m¹ represents independently for each occurrence 0, 1, 2, 3, 4, 5, 6, 7,or 8; Y represents independently for each occurrence an alkyl diradical,cycloalkyl diradical, heteroalkyl diradical, heterocycloalkyl diradical,alkenyl diradical, alkynyl diradical, aryl diradical, heteroaryldiradical, aralkyl diradical, heteroaralkyl diradical,—X²C(O)X²[C(R⁵)₂]_(v)X²—, —X²C(NR⁶)X²[C(R⁵)₂]_(v)X²—,—X²C(S)X²[C(R⁵)₂]_(v)X²—, —X²C(O)X²[C(R⁵)₂]_(v)X²C(O)X²—,

 —[C(R⁵)₂]_(t)N(R⁶)O[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)N(R⁶)N(R⁶)O[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)N(R⁷)C(O)[C(R⁵)₂]_(t)—, —[C(R⁵)₂]_(t)N(R)CO₂[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)N(R⁷)C(S)[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)N(R⁷)C(S)O[C(R⁵)₂]_(t)—, —[C(R⁵)₂]_(t)OC(O)S[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)SN(R⁷)CO₂[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)OSi(R⁸)₂O[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)OSO₂N(R⁷)[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)N(R⁷)SO₂N(R⁷)[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)SO₂N(morpholino)-[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)SO₂N(R⁷)[C(R⁵)₂]_(t)—, —[C(R¹)₂]_(t)S[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)OSO₂[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)S[C(R⁵)₂]_(y)O[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)O[C(R⁵)₂]_(y)O[C(R⁵)₂]_(t)—, —[C(R⁵)₂]_(t)O[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)N(R⁷)[C(R⁵)₂]_(t)—, —[C(R⁵)₂]_(t)C═NO[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)C(O)C(R⁵)═C(R⁵)[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)C(R⁵)═C(R⁵)[C(R⁵)₂]_(t)—, or—[C(R⁵)₂]_(t)X²C(O)X²[C(R⁵)₂]_(t)—; X² represents independently for eachoccurrence a bond, O, or N(R⁶); Z¹ represents independently for eachoccurrence O, S, or N(R⁸); Z² represents independently for eachoccurrence alkyl, aryl, aralkyl, B(R⁹)₃, —OM, —Oalkyl, —Oaryl,—Oaralkyl, —SM, —Salkyl, —Saryl, —Saralkyl, —[C(R⁵)₂]_(m)N(R⁶)₂,—N(R¹⁰)R¹¹, —N(R¹⁹)(C(R¹⁹)₂)_(m)N(R¹⁹)₂, —N(R⁷)C(O)R⁸, H, —OC(O)R⁸,—CO₂R⁸, F, Se, —SeR⁸, —(C(R¹⁹)₂)_(m)OR¹⁹, —(C(R¹⁹)₂)_(m)SR¹⁹,—N(R¹⁹)(C(R¹⁹)₂)_(m)OR¹⁹, —N(R¹⁹)(C(R¹⁹)₂)_(m)SR¹⁹,—N(R¹⁹)(C(R¹⁹)₂)_(m)N(R¹⁹)C(O)alkyl, —(C(R¹⁹)₂)_(m)N(R¹⁹)C(O)alkyl, or-A⁸-[A⁹-(A⁵)_(w)]_(y); R¹⁰ and R¹¹ are independently H, alkyl, or aryl;or R¹⁰ and R¹¹ taken together form a 3-, 4-, 5-, 6-, or 7-member ring;R¹² represents independently for each occurrence H, alkyl, or—NHCH₂CH═CH₂; t represents independently for each occurrence 0, 1, 2, 3,or 4; v represents independently for each occurrence 0, 1, 2, 3, 4, 5,6, 7, or 8; A⁵ represents independently for each occurrence aryl,aralkyl, or the radical of a steroid, bile acid, lipid, folic acid,pyridoxal, B12, riboflavin, biotin, polycyclic compound, crown ether,intercalator, cleaver molecule, protein-binding agent, carbohydrate, oran optionally substituted saturated 5-membered ring; A⁶ representsindependently for each occurrence a bond, alkyl diradical, heteroalkyldiradical, alkenyl diradical, aminoalkyl, —C(O)—, —S(O)—, —S(O)₂—, or isrepresented by formula:

Z³ represents independently for each occurrence O or S; Z⁴ representsindependently for each occurrence —OM, —Oalkyl, —Oaryl, —Oaralkyl, —SM,—Salkyl, —Saryl, —Saralkyl, —N(R¹⁰)R¹¹, —[C(R⁵)₂]N(R⁶)₂,—N(R¹⁹)(C(R¹⁹)₂)_(m)N(R¹⁹)₂, —(C(R¹⁹)₂)_(m)OR¹⁹, —(C(R¹⁹)₂)_(m)SR¹⁹,—N(R¹⁹)(C(R¹⁹)₂)_(m)OR¹⁹, —N(R¹⁹)(C(R¹⁹)₂)_(m)SR¹⁹,—N(R¹⁹)(C(R¹⁹)₂)_(m)N(R¹⁹)C(O)alkyl, —(C(R¹⁹)₂)_(m)N(R¹⁹)C(O)alkyl,aryl, or alkyl; R¹³ represent independently for each occurrence H,alkyl, cycloalkyl, heteroalkyl, aryl, aralkyl, acyl, silyl, or B³; R¹⁴represents independently for each occurrence alkyl, aryl, aralkyl, acyl,or silyl; R¹⁵ represents independently for each occurrence hydrogen,alkyl, aryl, aralkyl, acyl, alkylsulfonyl, alkylsulfoxide, arylsulfonyl,arylsulfoxide, or silyl; R¹⁶ represents independently for eachoccurrence cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; B³ is abond between A⁶ and A⁷; B⁴ is a bond between A⁶ and O; n³ representsindependently for each occurrence an integer in the range of 1-15,inclusive; n⁴ represents independently for each occurrence 1, 2, 3, 4,or 5 in accord with the rules of valence; A⁷ represents independentlyfor each occurrence a bond, alkyl diradical, heteroalkyl diradical,—C(O)—, —S(O)—, —S(O)₂—, B³C(R)₂B⁵, B³C(R)(B⁵)₂, B³C(B⁵)₃, B³N(R)(B⁵),B³N(B⁵)₂, or has the formula:

p represents independently for each occurrence 1, 2, 3, or 4; B⁵ is abond between A⁵ and A⁷; A⁸ is a bond, alkyl diradical, heteroalkyldiradical, alkenyl diradical, aminoalkyl, or is represented by formula:

R¹⁷ represent independently for each occurrence H, alkyl, cycloalkyl,heteroalkyl, aryl, aralkyl, acyl, silyl, or B⁶; R¹⁸ representsindependently for each occurrence H, halogen, alkyl, alkoxyl, —N(R⁶)₂,—CN, —[C(R⁵)₂]_(v)C(R⁵)═C(R)₂; R¹⁹ represents independently for eachoccurrence H or alkyl; B⁶ is a bond between A⁸ and A⁹; B⁷ is a bondbetween A³ and P; A⁹ is a bond, alkyl diradical, heteroalkyl diradical,—C(O)—, —S(O)—, —S(O)₂—, B⁶C(R)₂B⁸, B⁶C(R)(B⁸)₂, B⁶C(B⁸)₃, B⁶N(R)(B⁸),B⁶N(B⁸)₂, or has the formula:

B⁸ is a bond between A⁵ and A⁹; and provided that at least one instanceof Y is not


8. The compound of claim 7, wherein A⁵ is represented by formula III:

wherein R^(1-III), R^(2-III), and R^(3-III) represent independently foreach occurrence H, halogen, amino, hydroxyl, alkyl, alkoxyl, aminoalkyl,alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, thiol,thioalkyl, silyl, nitro, nitrile, acyl, acylamino, —COR, or —CO₂R. 9.The compound of claim 8, wherein R^(1-III) is methyl, R^(2-III) is H,and R^(3-III) is methoxy.
 10. The compound of claim 7, wherein A⁵ isrepresented by formula IV:

wherein R^(1-IV), R^(2-IV), and R^(3-IV) represent independently foreach occurrence H, halogen, amino, hydroxyl, alkyl, alkoxyl, aminoalkyl,alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, thiol,thioalkyl, silyl, nitro, nitrile, acyl, acylamino, —COR, or —CO₂R. 11.The compound of claim 10, wherein R^(1-IV) is methyl, R^(2-IV) is H, andR^(3-IV) is isobutyl.
 12. The compound of claim 10, wherein R³ and R⁴represent independently for each occurrence —NH₂, —N(H)CH₃, or —N(CH₃)₂.13. A single-stranded oligonucleotide represented by formula V:

wherein X¹ is H, —P(O)(OM)₂, —P(O)(OM)-O—P(O)(OM)₂, —P(O)(Oalkyl)₂,—P(O)(Oalkyl)-O—P(O)(Oalkyl)₂, or -A⁶-[A⁷-(A⁵)_(w)]_(y); M representsindependently for each occurrence an alkali metal or a transition metalwith an overall charge of +1; R¹ and R⁵ represent independently for eachoccurrence H, alkyl, or halogen; R² and R³ represent independently foreach occurrence H, OH, F, —Oalkyl, —Oallyl, —O(C(R¹⁹)₂)_(k)OR¹⁹,—O(C(R¹⁹)₂)_(k)SR¹⁹, —O(C(R¹⁹)₂)_(k)N(R¹⁹)₂, —O(C(R¹⁹)₂)_(k)C(O)N(R¹⁹)₂,—N(R¹⁹)₂, —S(C₁-C₆)alkyl, —O(C(R¹⁹)₂)_(k)O(C₁-C₆)alkyl,—O(C(R¹⁹)₂)_(k)S(C₁-C₆)alkyl,—O(C(R¹⁹)₂)_(k)O(C(R¹⁹)₂)_(k)N((C₁-C₆)alkyl)₂,—O(C(R¹⁹)₂)_(k)ON((C₁-C₆)alkyl)₂, or —O-A⁶-[A⁷-(A⁵)_(w)]_(y); R⁴represents independently for each occurrence H, OH, F, —Oalkyl, —Oallyl,—O(C(R¹⁹)₂)_(k)OR¹⁹, —O(C(R¹⁹)₂)_(k)SR¹⁹, —O(C(R¹⁹)₂)_(k)N(R¹⁹)₂,—O(C(R¹⁹)₂)_(k)C(O)N(R¹⁹)₂, —N(R¹⁹)₂, —S(C₁-C₆)alkyl,—O(C(R¹⁹)₂)_(k)O(C₁-C₆)alkyl, —O(C(R¹⁹)₂)_(k)S(C₁-C₆)alkyl,—O(C(R¹⁹)₂)_(k)O(C(R¹⁹)₂)_(k)N((C₁-C₆)alkyl)₂, or—O(C(R¹⁹)₂)_(k)ON((C₁-C₆)alkyl)₂; R⁶, R⁷, and R⁹ represent independentlyfor each occurrence H, alkyl, aryl, or aralkyl; R⁸ representsindependently for each occurrence alkyl, aryl, or aralkyl; k representsindependently for each occurrence 1, 2, 3, or 4; n¹ is 1, 2, or 3; n² isan integer in the range of about 15-28, inclusive; w representsindependently for each occurrence 1, 2, or 3 in accord with the rules ofvalence; x represents independently for each occurrence 0, 1, 2, or 3; yrepresents independently for each occurrence 1, 2, 3, 4, or 5 in accordwith the rules of valence; A¹ represents independently for eachoccurrence:

A² represents independently for each occurrence:

A³ represents independently for each occurrence

A⁴ represents independently for each occurrence a bond, alkyl diradical,heteroalkyl diradical, alkenyl diradical, alkynyl diradical,alkylalkynyl diradical, aminoalkyl diradical, thioether, —C(O)—, —S(O)—,—S(O)₂—, B¹C(R)₂B², B¹C(R)(B²)₂, B¹C(B²)₃, B¹N(R)(B²), B¹N(B²)₂, or hasthe formula:

B¹ is a bond between A³ and A⁴ B² is a bond between A⁴ and A⁵; Rrepresents independently for each occurrence hydrogen or alkyl; mrepresents independently for each occurrence 1, 2, 3, 4, 5, 6, 7, or 8;m¹ represents independently for each occurrence 0, 1, 2, 3, 4, 5, 6, 7,or 8; Y represents independently for each occurrence an alkyl diradical,cycloalkyl diradical, heteroalkyl diradical, heterocycloalkyl diradical,alkenyl diradical, alkynyl diradical, aryl diradical, heteroaryldiradical, aralkyl diradical, heteroaralkyl diradical,—X²C(O)X²[C(R⁵)₂]_(v)X²—, —X²C(NR⁶)X²[C(R⁵)₂]_(v)X²—,—X²C(S)X²[C(R⁵)₂]_(v)X²—, —X²C(O)X²[C(R⁵)₂]_(v)X²C(O)X²—,

 —[C(R⁵)₂]_(t)N(R⁶)O[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)N(R⁶)N(R⁶)O[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)N(R⁷)C(O)[C(R⁵)₂]_(t)—, —[C(R⁵)₂]_(t)N(R)CO₂[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)N(R⁷)C(S)[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)N(R⁷)C(S)O[C(R⁵)₂]_(t)—, —[C(R⁵)₂]_(t)OC(O)S[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)SN(R⁷)CO₂[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)OSi(R⁸)₂O[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)OSO₂N(R⁷)[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)N(R⁷)SO₂N(R⁷)[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)SO₂N(morpholino)-[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)SO₂N(R⁷)[C(R⁵)₂]_(t)—, —[C(R¹)₂]_(t)S[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)OSO₂[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)S[C(R⁵)₂]_(y)O[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)O[C(R⁵)₂]_(y)O[C(R⁵)₂]_(t)—, —[C(R⁵)₂]_(t)O[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)N(R⁷)[C(R⁵)₂]_(t)—, —[C(R⁵)₂]_(t)C═NO[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)C(O)C(R⁵)═C(R⁵)[C(R⁵)₂]_(t)—,—[C(R⁵)₂]_(t)C(R⁵)═C(R⁵)[C(R⁵)₂]_(t)—, or—[C(R⁵)₂]_(t)X²C(O)X²[C(R⁵)₂]_(t)—; X² represents independently for eachoccurrence a bond, O, or N(R⁶); Z¹ represents independently for eachoccurrence O, S, or N(R⁸); Z² represents independently for eachoccurrence alkyl, aryl, aralkyl, B(R⁹)₃, —OM, —Oalkyl, —Oaryl,—Oaralkyl, —SM, —Salkyl, —Saryl, —Saralkyl, —[C(R⁵)₂]_(m)N(R⁶)₂,—N(R¹⁰)R¹¹, —N(R¹⁹)(C(R¹⁹)₂)_(m)N(R¹⁹)₂, —N(R⁷)C(O)R⁸, H, —OC(O)R⁸,—CO₂R⁸, F, Se, —SeR⁸, —(C(R¹⁹)₂)_(m)OR¹⁹, —(C(R¹⁹)₂)_(m)SR¹⁹,—N(R¹⁹)(C(R¹⁹)₂)_(m)OR¹⁹, —N(R¹⁹)(C(R¹⁹)₂)_(m)SR¹⁹,—N(R¹⁹)(C(R¹⁹)₂)_(m)N(R¹⁹)C(O)alkyl, —(C(R¹⁹)₂)_(m)N(R¹⁹)C(O)alkyl, or-A⁸-[A⁹-(A⁵)_(w)]_(y); R¹⁰ and R¹¹ are independently H, alkyl, or aryl;or R¹⁰ and R¹¹ taken together form a 3-, 4-, 5-, 6-, or 7-member ring;R¹² represents independently for each occurrence H, alkyl, or—NHCH₂CH═CH₂; t represents independently for each occurrence 0, 1, 2, 3,or 4; v represents independently for each occurrence 0, 1, 2, 3, 4, 5,6, 7, or 8; A⁵ represents independently for each occurrence aryl,aralkyl, or the radical of a steroid, bile acid, lipid, folic acid,pyridoxal, B12, riboflavin, biotin, polycyclic compound, crown ether,intercalator, cleaver molecule, protein-binding agent, carbohydrate, oran optionally substituted saturated 5-membered ring; A⁶ representsindependently for each occurrence a bond, alkyl diradical, heteroalkyldiradical, alkenyl diradical, aminoalkyl, —C(O)—, —S(O)—, —S(O)₂—, or isrepresented by formula:

Z³ represents independently for each occurrence O or S; Z⁴ representsindependently for each occurrence —OM, —Oalkyl, —Oaryl, —Oaralkyl, —SM,—Salkyl, —Saryl, —Saralkyl, —N(R¹⁰)R¹¹, —[C(R⁵)₂]_(m)N(R⁶)₂,—N(R¹⁹)(C(R¹⁹)₂)_(m)N(R¹⁹)₂, —(C(R¹⁹)₂)_(m)OR¹⁹, —(C(R¹⁹)₂)_(m)SR¹⁹,—N(R¹⁹)(C(R¹⁹)₂)_(m)OR¹⁹, —N(R¹⁹)(C(R¹⁹)₂)_(m)SR¹⁹,—N(R¹⁹)C(R¹⁹)₂)_(m)N(R¹⁹)(C(O)alkyl, —(C(R¹⁹)₂)_(m)N(R¹⁹)C(O)alkyl,aryl, or alkyl; R¹³ represent independently for each occurrence H,alkyl, cycloalkyl, heteroalkyl, aryl, aralkyl, acyl, silyl, or B³; R¹⁴represents independently for each occurrence alkyl, aryl, aralkyl, acyl,or silyl; R¹⁵ represents independently for each occurrence hydrogen,alkyl, aryl, aralkyl, acyl, alkylsulfonyl, alkylsulfoxide, arylsulfonyl,arylsulfoxide, or silyl; R¹⁶ represents independently for eachoccurrence cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; B³ is abond between A⁶ and A⁷; B⁴ is a bond between A⁶ and O; n³ representsindependently for each occurrence an integer in the range of 1-15,inclusive; n⁴ represents independently for each occurrence 1, 2, 3, 4,or 5 in accord with the rules of valence; A⁷ represents independentlyfor each occurrence a bond, alkyl diradical, heteroalkyl diradical,—C(O)—, —S(O)—, —S(O)₂—, B³C(R)₂B⁵, B³C(R)(B⁵)₂, B³C(B⁵)₃, B³N(R)(B⁵),B³N(B⁵)₂, or has the formula:

p represents independently for each occurrence 1, 2, 3, or 4; B⁵ is abond between A⁵ and A⁷; A⁸ is a bond, alkyl diradical, heteroalkyldiradical, alkenyl diradical, aminoalkyl, or is represented by formula:

R¹⁷ represent independently for each occurrence H, alkyl, cycloalkyl,heteroalkyl, aryl, aralkyl, acyl, silyl, or B⁶; R¹⁸ representsindependently for each occurrence H, halogen, alkyl, alkoxyl, —N(R⁶)₂,—CN, —[C(R⁵)₂]_(v)C(R⁵)═C(R⁵)₂; R¹⁹ represents independently for eachoccurrence H or alkyl; B⁶ is a bond between A⁸ and A⁹; B⁷ is a bondbetween A⁸ and P; A⁹ is a bond, alkyl diradical, heteroalkyl diradical,—C(O)—, —S(O)—, —S(O)₂—, B⁶C(R)₂B⁸, B⁶C(R)(B⁸)₂, B⁶C(B⁸)₃, B⁶N(R)(B⁸),B⁶N(B⁸)₂, or has the formula:

B⁸ is a bond between A⁵ and A⁹; and provided that at least one instanceof Y is not


14. The compound of claim 13, wherein A⁵ is represented by formula VI:

wherein R^(1-VI), R^(2-VI), and R^(3-VI) represent independently foreach occurrence H, halogen, amino, hydroxyl, alkyl, alkoxyl, aminoalkyl,alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, thiol,thioalkyl, silyl, nitro, nitrile, acyl, acylamino, —COR, or —CO₂R. 15.The compound of claim 14, wherein R^(1-VI) is methyl, R^(2-VI) is H, andR^(3-VI) is methoxy.
 16. The compound of claim 13, wherein A⁵ isrepresented by formula VII:

wherein R^(1-VII), R^(2-VII), and R^(3-VII) represent independently foreach occurrence H, halogen, amino, hydroxyl, alkyl, alkoxyl, aminoalkyl,alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, thiol,thioalkyl, silyl, nitro, nitrile, acyl, acylamino, —COR, or —CO₂R. 17.The compound of claim 16, wherein R^(1-VII) is methyl, R^(2-VII) is H,and R^(3-VII) is isobutyl.
 18. The compound of claim 16, wherein R³ andR⁴ represent independently for each occurrence —NH₂, —N(H)CH₃, or—N(CH₃)₂.
 19. A method of treating a patient suffering from a maladyselected from the group consisting of unwanted cell proliferation,arthritis, retinal neovascularization, viral infection, bacterialinfection, amoebic infection, parasitic infection, fungal infection,unwanted immune response, asthma, lupus, multiple sclerosis, diabetes,acute pain, chronic pain, neurological disease, and a disordercharacterized by loss of heterozygosity; comprising the step of:administering to a patient in need thereof a therapeutically effectiveamount of a oligonucleotide according to claim 7 or
 13. 20. A method ofgene-silencing, comprising the steps of: administering a therapeuticallyeffective amount of an oligonucleotide according to claim 7 or 13 to amammalian cell to silence a gene promoting unwanted cell proliferation,growth factor gene, growth factor receptor gene, a kinase gene, a geneencoding a G protein superfamily molecule, a gene encoding atranscription factor, a gene which mediates angiogenesis, a viral geneof a cellular gene which mediates viral function, a gene of a bacterialpathogen, a gene of an amoebic pathogen, a gene of a parasitic pathogen,a gene of a fungal pathogen, a gene which mediates an unwanted immuneresponse, a gene which mediates the processing of pain, a gene whichmediates a neurological disease, an allene gene found in cellscharacterized by loss of heterozygosity, or one allege gene of apolymorphic gene.
 21. A method of gene-silencing, comprising the stepsof: administering a therapeutically effective amount of anoligonucleotide according to claim 7 or 13 to a mammalian cell tosilence a PDGF beta gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RASgene, MEKK gene, JNK gene, RAF gene, Erk1/2 gene, PCNA(p21) gene, MYBgene, JUN gene, FOS gene, BCL-2 gene, Cyclin D gene, VEGF gene, EGFRgene, Cyclin A gene, Cyclin E gene, WNT-1 gene, beta-catenin gene, c-METgene, PKC gene, NFKB gene, STAT3 gene, survivin gene, Her2/Neu gene,topoisomerase I gene, topoisomerase II alpha gene, mutations in the p73gene, mutations in the p21 (WAF1/CIP1) gene, mutations in the p27(KIP1)gene, mutations in the PPM1D gene, mutations in the RAS gene, mutationsin the caveolin I gene, mutations in the MIB I gene, mutations in theMTAI gene, mutations in the M68 gene, mutations in tumor suppressorgenes, mutations in the p53 tumor suppressor gene, mutations in the p53family member DN-p63, mutations in the pRb tumor suppressor gene,mutations in the APC1 tumor suppressor gene, mutations in the BRCA1tumor suppressor gene, mutations in the PTEN tumor suppressor gene, mLLfusion gene, BCR/ABL fusion gene, TEL/AML1 fusion gene, EWS/FLI1 fusiongene, TLS/FUS1 fusion gene, PAX3/FKHR fusion gene, AML1/ETO fusion gene,alpha v-integrin gene, Flt-1 receptor gene, tubulin gene, HumanPapilloma Virus gene, a gene required for Human Papilloma Virusreplication, Human Immunodeficiency Virus gene, a gene required forHuman Immunodeficiency Virus replication, Hepatitis A Virus gene, a generequired for Hepatitis A Virus replication, Hepatitis B Virus gene, agene required for Hepatitis B Virus replication, Hepatitis C Virus gene,a gene required for Hepatitis C Virus replication, Hepatitis D Virusgene, a gene required for Hepatitis D Virus replication, Hepatitis EVirus gene, a gene required for Hepatitis E Virus replication, HepatitisF Virus gene, a gene required for Hepatitis F Virus replication,Hepatitis G Virus gene, a gene required for Hepatitis G Virusreplication, Hepatitis H Virus gene, a gene required for Hepatitis HVirus replication, Respiratory Syncytial Virus gene, a gene that isrequired for Respiratory Syncytial Virus replication, Herpes SimplexVirus gene, a gene that is required for Herpes Simplex Virusreplication, herpes Cytomegalovirus gene, a gene that is required forherpes Cytomegalovirus replication, herpes Epstein Barr Virus gene, agene that is required for herpes Epstein Barr Virus replication,Kaposi's Sarcoma-associated Herpes Virus gene, a gene that is requiredfor Kaposi's Sarcoma-associated Herpes Virus replication, JC Virus gene,human gene that is required for JC Virus replication, myxovirus gene, agene that is required for myxovirus gene replication, rhinovirus gene, agene that is required for rhinovirus replication, coronavirus gene, agene that is required for coronavirus replication, West Nile Virus gene,a gene that is required for West Nile Virus replication, St. LouisEncephalitis gene, a gene that is required for St. Louis Encephalitisreplication, Tick-borne encephalitis virus gene, a gene that is requiredfor Tick-borne encephalitis virus replication, Murray Valleyencephalitis virus gene, a gene that is required for Murray Valleyencephalitis virus replication, dengue virus gene, a gene that isrequired for dengue virus gene replication, Simian Virus 40 gene, a genethat is required for Simian Virus 40 replication, Human T CellLymphotropic Virus gene, a gene that is required for Human T CellLymphotropic Virus replication, Moloney-Murine Leukemia Virus gene, agene that is required for Moloney-Murine Leukemia Virus replication,encephalomyocarditis virus gene, a gene that is required forencephalomyocarditis virus replication, measles virus gene, a gene thatis required for measles virus replication, Vericella zoster virus gene,a gene that is required for Vericella zoster virus replication,adenovirus gene, a gene that is required for adenovirus replication,yellow fever virus gene, a gene that is required for yellow fever virusreplication, poliovirus gene, a gene that is required for poliovirusreplication, poxvirus gene, a gene that is required for poxvirusreplication, plasmodium gene, a gene that is required for plasmodiumgene replication, Mycobacterium ulcerans gene, a gene that is requiredfor Mycobacterium ulcerans replication, Mycobacterium tuberculosis gene,a gene that is required for Mycobacterium tuberculosis replication,Mycobacterium leprae gene, a gene that is required for Mycobacteriumleprae replication, Staphylococcus aureus gene, a gene that is requiredfor Staphylococcus aureus replication, Streptococcus pneumoniae gene, agene that is required for Streptococcus pneumoniae replication,Streptococcus pyogenes gene, a gene that is required for Streptococcuspyogenes replication, Chlamydia pneumoniae gene, a gene that is requiredfor Chlamydia pneumoniae replication, Mycoplasma pneumoniae gene, a genethat is required for Mycoplasma pneumoniae replication, an integringene, a selectin gene, complement system gene, chemokine gene, chemokinereceptor gene, GCSF gene, Gro1 gene, Gro2 gene, Gro3 gene, PF4 gene, MIGgene, Pro-Platelet Basic Protein gene, MIP-1I gene, MIP-1J gene, RANTESgene, MCP-1 gene, MCP-2 gene, MCP-3 gene, CMBKR1 gene, CMBKR2 gene,CMBKR3 gene, CMBKR5v, AIF-1 gene, I-309 gene, a gene to a component ofan ion channel, a gene to a neurotransmitter receptor, a gene to aneurotransmitter ligand, amyloid-family gene, presenilin gene, HD gene,DRPLA gene, SCA1 gene, SCA2 gene, MJD1 gene, CACNL1A4 gene, SCA7 gene,SCA8 gene, allele gene found in LOH cells, or one allele gene of apolymorphic gene.
 22. A method of gene-silencing, comprising the stepsof: administering a therapeutically effective amount of anoligonucleotide according to claim 7 or 13 to a mammal to silence a genepromoting unwanted cell proliferation, growth factor or growth factorreceptor gene, a kinase gene, a gene encoding a G protein superfamilymolecule, a gene encoding a transcription factor, a gene which mediatesangiogenesis, a viral gene of a cellular gene which mediates viralfunction, a gene of a bacterial pathogen, a gene of an amoebic pathogen,a gene of a parasitic pathogen, a gene of a fungal pathogen, a genewhich mediates an unwanted immune response, a gene which mediates theprocessing of pain, a gene which mediates a neurological disease, anallene gene found in cells characterized by loss of heterozygosity, orone allege gene of a polymorphic gene.
 23. A method of gene-silencing,comprising the steps of: administering a therapeutically effectiveamount of an oligonucleotide according to claim 7 or 13 to a mammal tosilence a PDGF beta gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RASgene, MEKK gene, JNK gene, RAF gene, Erk1/2 gene, PCNA(p21) gene, MYBgene, JUN gene, FOS gene, BCL-2 gene, Cyclin D gene, VEGF gene, EGFRgene, Cyclin A gene, Cyclin E gene, WNT-1 gene, beta-catenin gene, c-METgene, PKC gene, NFKB gene, STAT3 gene, survivin gene, Her2/Neu gene,topoisomerase I gene, topoisomerase II alpha gene, mutations in the p73gene, mutations in the p21(WAF1/CIP1) gene, mutations in the p27(KIP1)gene, mutations in the PPM1D gene, mutations in the RAS gene, mutationsin the caveolin I gene, mutations in the MIB I gene, mutations in theMTAI gene, mutations in the M68 gene, mutations in tumor suppressorgenes, mutations in the p53 tumor suppressor gene, mutations in the p53family member DN-p63, mutations in the pRb tumor suppressor gene,mutations in the APC1 tumor suppressor gene, mutations in the BRCA1tumor suppressor gene, mutations in the PTEN tumor suppressor gene, mLLfusion gene, BCR/ABL fusion gene, TEL/AML1 fusion gene, EWS/FLI1 fusiongene, TLS/FUS1 fusion gene, PAX3/FKHR fusion gene, AML1/ETO fusion gene,alpha v-integrin gene, Flt-1 receptor gene, tubulin gene, HumanPapilloma Virus gene, a gene required for Human Papilloma Virusreplication, Human Immunodeficiency Virus gene, a gene required forHuman Immunodeficiency Virus replication, Hepatitis A Virus gene, a generequired for Hepatitis A Virus replication, Hepatitis B Virus gene, agene required for Hepatitis B Virus replication, Hepatitis C Virus gene,a gene required for Hepatitis C Virus replication, Hepatitis D Virusgene, a gene required for Hepatitis D Virus replication, Hepatitis EVirus gene, a gene required for Hepatitis E Virus replication, HepatitisF Virus gene, a gene required for Hepatitis F Virus replication,Hepatitis G Virus gene, a gene required for Hepatitis G Virusreplication, Hepatitis H Virus gene, a gene required for Hepatitis HVirus replication, Respiratory Syncytial Virus gene, a gene that isrequired for Respiratory Syncytial Virus replication, Herpes SimplexVirus gene, a gene that is required for Herpes Simplex Virusreplication, herpes Cytomegalovirus gene, a gene that is required forherpes Cytomegalovirus replication, herpes Epstein Barr Virus gene, agene that is required for herpes Epstein Barr Virus replication,Kaposi's Sarcoma-associated Herpes Virus gene, a gene that is requiredfor Kaposi's Sarcoma-associated Herpes Virus replication, JC Virus gene,human gene that is required for JC Virus replication, myxovirus gene, agene that is required for myxovirus gene replication, rhinovirus gene, agene that is required for rhinovirus replication, coronavirus gene, agene that is required for coronavirus replication, West Nile Virus gene,a gene that is required for West Nile Virus replication, St. LouisEncephalitis gene, a gene that is required for St. Louis Encephalitisreplication, Tick-borne encephalitis virus gene, a gene that is requiredfor Tick-borne encephalitis virus replication, Murray Valleyencephalitis virus gene, a gene that is required for Murray Valleyencephalitis virus replication, dengue virus gene, a gene that isrequired for dengue virus gene replication, Simian Virus 40 gene, a genethat is required for Simian Virus 40 replication, Human T CellLymphotropic Virus gene, a gene that is required for Human T CellLymphotropic Virus replication, Moloney-Murine Leukemia Virus gene, agene that is required for Moloney-Murine Leukemia Virus replication,encephalomyocarditis virus gene, a gene that is required forencephalomyocarditis virus replication, measles virus gene, a gene thatis required for measles virus replication, Vericella zoster virus gene,a gene that is required for Vericella zoster virus replication,adenovirus gene, a gene that is required for adenovirus replication,yellow fever virus gene, a gene that is required for yellow fever virusreplication, poliovirus gene, a gene that is required for poliovirusreplication, poxvirus gene, a gene that is required for poxvirusreplication, plasmodium gene, a gene that is required for plasmodiumgene replication, Mycobacterium ulcerans gene, a gene that is requiredfor Mycobacterium ulcerans replication, Mycobacterium tuberculosis gene,a gene that is required for Mycobacterium tuberculosis replication,Mycobacterium leprae gene, a gene that is required for Mycobacteriumleprae replication, Staphylococcus aureus gene, a gene that is requiredfor Staphylococcus aureus replication, Streptococcus pneumoniae gene, agene that is required for Streptococcus pneumoniae replication,Streptococcus pyogenes gene, a gene that is required for Streptococcuspyogenes replication, Chlamydia pneumoniae gene, a gene that is requiredfor Chlamydia pneumoniae replication, Mycoplasma pneumoniae gene, a genethat is required for Mycoplasma pneumoniae replication, an integringene, a selectin gene, complement system gene, chemokine gene, chemokinereceptor gene, GCSF gene, Gro1 gene, Gro2 gene, Gro3 gene, PF4 gene, MIGgene, Pro-Platelet Basic Protein gene, MIP-1I gene, MIP-1J gene, RANTESgene, MCP-1 gene, MCP-2 gene, MCP-3 gene, CMBKR1 gene, CMBKR2 gene,CMBKR3 gene, CMBKR5v, AIF-1 gene, I-309 gene, a gene to a component ofan ion channel, a gene to a neurotransmitter receptor, a gene to aneurotransmitter ligand, amyloid-family gene, presenilin gene, HD gene,DRPLA gene, SCA1 gene, SCA2 gene, MJD1 gene, CACNL1A4 gene, SCA7 gene,SCA8 gene, allele gene found in LOH cells, or one allele gene of apolymorphic gene.